AU711016B2 - High level expression, purification and refolding of the Neisseria meningitidis outer membrane group B porin proteins - Google Patents

Description

I P00011 Regulation 3.2

AUSTRALIA

Patents Act, 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANTS: THE ROCKEFELLER UNIVERSITY AND NORTH AMERICAN VACCINE, INC.

ACTUAL INVENTORS: ADDRESS FOR SERVICE: MILAN S BLAKE; JOSEPH Y TAI; HUILIN L QI; SHU-MEI LIANG; LUCJAN J J HRONOWSKI and JEFFREY K PULLEN Peter Maxwell Associates Level 6 Pitt Street SYDNEY NSW 2000 HIGH LEVEL EXPRESSION, PURIFICATION AND REFOLDING OF THE NEISSERIA MENINGITIDIS OUTER MEMBRANE GROUP B PORIN PROTEINS INVENTION TITLE: The following statement is a full description of this invention including the best method of performing it known to me:- IP AUSTRALIA

RECEIVED

14 JUL 1998

SYDNEY

HIGH LEVEL EXPRESSION, PURIFICATION AND REFOLDING OF THE NEISSERIA MENINGITIDIS OUTER MEMBRANE GROUP B PORIN PROTEINS Background of the Invention Field of the Invention The present invention is in the field of recombinant genetics, protein expression, and vaccines. The present invention relates, in particular, to a method of expressing in a recombinant host an outer membrane group B porin protein from Neisseria meningitidis. The invention also relates to a method of purification and refolding of the recombinant protein.

Background Information The outer membranes of Neisseria species much like other Gram negative bacteria are semi-permeable membranes which allow free flow access and escape of small molecular weight substances to and from the 15 periplasmic space of these bacteria but retard molecules of larger size (Heasley, et al., "Reconstitution and characterization of the N.

gonorrhoeae outer membrane permeability barrier," in Genetics and Immunobiology of Neisseria gonorrhoeae, Danielsson and Normark, eds., University of Umea, Umea, pp. 12-15 (1980); Douglas, et al., FEMS 20 Microbiol. Lett. 12:305-309 (1981)). One of the mechanisms whereby this is accomplished is the inclusion within these membranes of proteins which have been collectively named porins. These proteins are made up of three identical polypeptide chains (Jones, et al., Infect. Immun. 30:773-780 (1980); McDade, Jr. and Johnston, J. Bacteriol. 141:1183-1191 (1980)) 25 and in their native trimer conformation, form water filled, voltagedependent channels within the outer membrane of the bacteria or other membranes to which they have been introduced (Lynch, et al., Biophys. J. 41:62 (1983); Lynch, et al., Biophys. J. 45:104-107 -2- (1984); Young, et al., Proc. Natl. Acad. Sci. USA 80:3831-3835 (1983); Mauro, et al., Proc. Natl. Acad. Sci. USA 85:1071-1075 (1988); Young, et al., Proc. Natl. Acad. Sci. USA 83:150-154 (1986)). Because of the relative abundance of these proteins within the outer membrane, these protein antigens have also been used to subgroup both Neisseria gonorrhoeae and Neisseria meningitidis into several serotypes for epidemiological purposes (Frasch, et al., Rev. Infect.

Dis. 7:504-510 (1985); Knapp, et al., "Overview of epidemiological and clinical applications of auxotype/serovar classification of Neisseria gonorrhoeae," The Pathogenic Neisseriae, Schoolnik, ed., American Society for Microbiology, Washington, pp. 6-12 (1985)). To date, many of these proteins from both gonococci and meningococci have been purified (Heckels, J. Gen. Microbiol. 99:333-341 (1977); James and Heckels, J. Immunol. Meth. 42:223-228 (1981); Judd, Anal. Biochem.

173:307-316 (1988); Blake and Gotschlich, Infect. Immun. 36:277-283 (1982); Wetzler, et al., J. Exp. Med. 168:1883-1897 (1988)), and cloned and sequenced (Gotschlich, et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); McGuinness, et al., J. Exp. Med. 171:1871-1882 (1990); Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 20 (1987); Feavers, et al., Infect. Immun. 60:3620-3629 (1992); t Murakami, etal., Infect. Immun. 57:2318-2323 (1989); Wolff and Stern, FEMS Microbiol. Lett. 83:179-186 (1991); Ward, et al., FEMS Microbiol. Lett. 73:283-289 (1992)).

The porin proteins were initially co-isolated with lipopolysaccharides. Consequently, the porin proteins have been termed "endotoxin-associated proteins" (Bjornson et al., Infect. Immun. 56:1602- 1607 (1988)). Studies on the wild type porins have reported that full assembly and oligomerization are not achieved unless LPS from the corresponding bacterial strain is present in the protein environment -3- (Holzenburg et al., Biochemistry 28:4187-4193 (1989); Sen and Nikaido, J. Biol. Chem. 266:11295-11300 (1991)).

The meningococcal porins have been subdivided into three major classifications which in antedated nomenclature were known as Class 1, 2, and 3 (Frasch, et al., Rev. Infect. Dis. 7:504-510 (1985)). Each meningococcus examined has contained one of the alleles for either a Class 2 porin gene or a Class 3 porin gene but not both (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)); Murakami, et al., Infect. Immun.

57:2318-2323 (1989)). The presence or absence of the Class 1 gene appears to be optional. Likewise, all probed gonococci contain only one porin gene with similarities to either the Class 2 or Class 3 allele (Gotschlich, et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987)).

N. gonorrhoeae appear to completely lack the Class 1 allele. The data from the genes that have been thus far sequenced would suggest that all neisserial porin proteins have at least 70% homology with each other with some variations on a basic theme (Feavers, et al., Infect. Immun.

i 60:3620-3629 (1992)). It has been suggested that much of the variation seen between these neisserial porin proteins is due to the immunological 20 pressures brought about by the invasion of these pathogenic organisms into their natural host, man. However, very little is known about how the Schanges in the porin protein sequence effect the functional activity of these proteins.

It has been previously reported that isolated gonococcal porins of the 25 Class 2 allelic type behave electrophysically somewhat differently than isolated gonococcal porins of the Class 3 type in lipid bilayer studies both Sin regards to their ion selectivity and voltage-dependence (Lynch, E.C., et al., Biophys. J. 41:62 (1983); Lynch, et al., Biophys. J. 45:104- 107 (1984)). Furthermore, the ability of the different porins to enter these _________IIIIIILIIII111 -4lipid bilayers from intact living bacteria seems to correlate not only with the porin type but also with the neisserial species from which they were donated (Lynch, et al., Biophys. J. 45:104-107 (1984)). It would seem that at least some of these functional attributes could be related to different areas within the protein sequence of the porin. One such functional area, previously identified within all gonococcal Class 2-like proteins, is the site of chymotrypsin cleavage. Upon chymotrypsin digestion, this class of porins lack the ability to respond to a voltage potential and close. Gonococcal Class 3-like porins as well as meningococcal porins lack this sequence and are thus not subject to chymotrypsin cleavage but nonetheless respond by closing to an applied voltage potential (Greco, "The formation of channels in lipid bilayers by gonococcal major outer membrane protein," thesis, The Rockefeller University, New York (1981); Greco, et al., Fed. Proc. 39:1813 (1980)).

The major impediment for such studies has been the ability to easily manipulate the porin genes by modem molecular techniques and obtain sufficient purified protein to carry out the biophysical characterizations of these altered porin proteins. It was early recognized that cloned neisserial 20 porin genes, when expressed in Escherichia coli, were lethal to the host E. coli (Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987); Carbonetti, et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988); Barlow, et al., Infect. Immun. 55:2734-2740 (1987)). Thus, many of these genes were cloned and sequenced as pieces of the whole S 25 gene or placed into low copy number plasmids under tight expression control (Carbonetti, et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 Under these conditions, even when the entire porin gene was expressed, very little protein accumulated that could be further purified and processed for characterization.

Another tack to this problem which has met with a modicum of success has been to clone the porin genes into a low copy, tightly controlled expression plasmid, introduce modifications to the porin gene, and then reintroduce the modified sequence back into Neisseria (Carbonetti, et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)). However, this has also been fraught with problems due to the elaborate restriction endonuclease system present in Neisseria, especially gonococci (Davies, Clin. Microbiol. Rev. 2:S78-S82 (1989)).

The present invention is directed to an approach to overcome these difficulties. The DNA sequence of the mature porin proteins, e.g. class 2 and class 3 as well as fusions thereof, may be amplified using the chromosome of the meningococcal bacteria as a template for the PCR reaction. The amplified porin sequences were ligated and cloned into an expression vector containing the T7 promoter. E. coli strain BL21 lysogenic for the DE3 lambda phage (Studier and Moffatt, J. Mol. Biol.

189:113-130 (1986)), modified to eliminate the ompA gene, was selected as one expression host for the pET-17b plasmid containing the porin gene.

Upon induction, large amounts of the meningococcal porin proteins accumulated within the E. coli without any obvious lethal effects to the host 20 bacterium. The expressed meningococcal porin proteins were extracted and processed through standard procedures and finally purified by molecular sieve chromatography and ion exchange chromatography. As judged by the •protein profile from the molecular sieve chromatography, the recombinant meningococcal porins eluted from the column as trimers. To be certain that no PCR artifacts had been introduced into the meningococcal porin genes to allow for such high expression, the inserted PorB gene sequence was determined. Inhibition ELISA assays were used to give further evidence that the expressed recombinant porin proteins had renatured into their natural antigenic and trimer conformation.

Summary of the Invention Points from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porin molecules correlate with these observed biophysical changes has been impeded by the ability to easily manipulate the cloned porin genes by modern molecular techniques and then subsequently obtain enough of the expressed modified porin protein to purify and apply to these biophysical functional assays. In this invention, the gene coding for a mature PorB protein, lacking the neisserial promoter and signal sequence, was cloned into the expression plasmid pET-17b and transformed into E. coli. Upon induction, large amounts of PorB protein was produced.

The expressed porin protein was then manipulated to regenerate its native trimer structure and was then purified. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. Thus, this sets the stage whereby the biophysical characterisation of these neisserial porin proteins can be examined in more detail.

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It is an object of the invention to provide a method of refolding a recombinantly produced outer membrane meningococcal group B porin protein or fusion protein thereof.

According to one aspect of the invention there is provided a method of refolding a recombinantly produced outer membrane meningococcal group B porin protein or fusion protein thereof, comprising: lysing transformed E. coli host cells capable of expressing the mature meningococcal group B porin protein or fusion protein thereof to release the protein as insoluble inclusion bodies; i washing said insoluble inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; suspending and dissolving said inclusion bodies in an aqueous solution of a denaturant; diluting said solution with a detergent, with the proviso that said detergent is not SDS; and passing said diluted solution through a gel filtration column; whereby folded, trimeric protein is obtained.

According to the invention there is also provided a vaccine 20 comprising the meningococcal group B porin protein and fusion protein, produced according to the above method, in an amount effective to elicit 29/7/99 8 protective antibodies in an animal to Neisseria meningitidis; and a pharmaceutically acceptable diluent, carrier, or excipient.

The invention further provides the above-described vaccine, wherein said meningococcal group B porin protein or fusion protein is conjugated to a Neisseria meningitidis capsular polysaccharide.

According to another aspect of the invention there is provided a method of obtaining a meningococcal group B porin protein or fusion protein-polysaccharide conjugate comprising: obtaining the refolded protein according to the method of claim 1; obtaining a Neisseria meningitidis capsular polysaccharide; and conjugating the protein to the polysaccharide of According to a further aspect of the invention there is provided a method of preventing bacterial meningitis in an animal comprising administering to the animal the meningococcal group B porin protein or fusion protein thereof produced according to the above-described method.

:'Brief Description of the Drawings Figure 1: A diagram showing the sequencing strategy of the PorB 20 gene. The PCR product described in Example 1 (Materials and Methods section) was ligated into the BamHI-Xhol site of the expression plasmid pET-17b. The initial double stranded primer extension sequencing was accomplished using oligonucleotide sequences directly upstream of the 29/7/99 9 BamHI site and just downstream of the Xhol site within the pET-1 7b plasmid. Additional sequence data was obtained by making numerous deletions in the 3' end of the gene, using exonuclease Ill/mung bean nuclease reactions. After religation and transformation back into E.coli, several clones were selected on size of insert and subsequently sequenced.

This sequencing was always from the 3' end of the gene using an oligonucleotide primer just downstream of the Bpul 1021 site.

Figure 2: A gel electrophoresis showing the products of the PCR reaction (electrophoresed in a 1% agarose using TAE buffer).

Figure 3 (panels and Panel SDS-PAGE analysis of whole cell lysates of E.coli hosting the control pET-17b plasmid without inserts and an E.coli clone harboring pET-17b plasmid containing an insert from the obtained PCR product described in the materials and methods section.

Both cultures were grown to an O.D. of 0.6 at 600 nm, IPTG added, and incubated at 370C for 2 hrs. 1.5 mls of each of the cultures -x

A--

2.

were removed, centrifuged, and the bacterial pellet solubilized in 100 gl of SDS-PAGE preparation buffer. Lane A shows the protein profile obtained with 10 pl from the control sample and Lanes B (5 xl) and C (10 pl) demonstrate the protein profile of the E. coli host expressing the PorB protein. Panel Western blot analysis of whole cell lysates of E. coli harboring the control pET-17b plasmid without insert after 2 hrs induction with IPTG, Lane A, 20 1 l and a corresponding E. coli clone containing a porB-pET-17b plasmid, Lane B, 5 pl; Lane C, 10 and Lane D, 20 pl.

The monoclonal antibody 4D11 was used as the primary antibody and the western blot developed as described. The pre-stained low molecular weight standards from BRL were used in each case.

Figure 4: The nucleotide sequence (SEQ ID NO. 1) and the translated amino acid sequence (SEQ ID NO. 2) of the mature PorB gene cloned into the expression plasmid pET-17b. The two nucleotides which differ from the previously published serotype 15 PorB are underlined.

Figure 5: A graph showing the Sephacryl S-300 column elution profile of both the wild type Class 3 protein isolated from the meningococcal strain 8765 and the recombinant Class 3 protein produced :by BL21(DE3) -AompAE. coli strain hosting the r3pET-17b [is this a typo 20 it does not appear in the Examples] plasmid as monitored by absorption at 280nm and SDS-PAGE analysis. The void volume of the column is indicated by the arrow. Fractions containing the meningococcal porin and recombinant porin as determined by SDS-PAGE are noted by the bar.

Figure 6: A graph showing the results of the inhibition ELISA 25 assays showing the ability of the homologous, wild type (wt) PorB to compete for reactive antibodies in six human immune sera. The arithmetic mean inhibition is shown by the bold line.

Figure 7: A graph showing the results of the inhibition ELISA assays showing the ability of the purified recombinant PorB protein to 11 20 2 25 compete for reactive antibodies in six human immune sera. The arithmetic mean inhibition is shown by the bold line.

Figure 8: A graph showing a comparison of these two mean inhibitions obtained with the wt and recombinant PorB protein.

Figure 9A and 9B: The nucleotide sequence (SEQ ID NO. 3) and the translated amino acid sequence (SEQ ID NO. 4) of the mature class II porin gene cloned into the expression plasmid pET-17b.

Figure 10A and 10B: The nucleotide sequence (SEQ ID NO. and the translated amino acid sequence (SEQ ID NO. 6) of the fusion class II porin gene cloned into the expression plasmid pET-17b.

Figure 11 (panels A and Panel A depicts the restriction map of the pET-17b plasmid. Panel B depicts the nucleotide sequence (SEQ ID NO. 7 AND SEQ ID NO. 9) between the BgII and Xhol sites of pET-17b.

The sequence provided by the plasmid is in normal print while the sequence inserted from the PCR product are identified in bold print. The amino acids (SEQ ID NO. 8 AND SEQ ID NO. 10) which are derived from the plasmid are in normal print while the amino acids from the insert are in bold. The arrows demarcate where the sequence begins to match the sequence in Figure 4 and when it ends.

Detailed Description of the Invention Unlike the porin proteins of E. coli and a few other gram negative bacteria, relatively little is known how changes in the primary sequence of porins from Neisseria effect their ion selectivity, voltage dependence, and other biophysical functions. Recently, the crystalline structure of two E. coli porins, OmpF and PhoE, were solved to 2.4A and respectively (Cowan, et al., Nature 358:727-733 (1992)). Both of these E. coli porins have been intensively studied owing to their unusual 12stability and ease with which molecular genetic manipulations could be accomplished. The data obtained for the genetics of these two porins correlated well with the crystalline structure. Although it has been shown in several studies, using monoclonal antibodies to select neisserial porins, that the surface topology of Neisseria closely resembles that of these two E. coli porins (van der Ley, et al., Infect. Immun. 59:2963-2971 (1991)), almost no information is available about how changes in amino acid sequences in specific areas of the neisserial porins effect their biophysical characteristics, as had been done with the E. coli porins (Cowan, et al., Nature 358:727-733 (1992)).

Two of the major problems impeding this research are: the inability to easily manipulate Neisseria genetically by modem molecular techniques and the inability to express sufficient quantities of neisserial porins in E. coli for further purification to obtain biophysical and biochemical characterization data. In fact, most of the DNA sequence data on gonococcal and meningococcal porins have been obtained by cloning overlapping pieces of the porin gene and then reconstructing the information to reveal the entire gene sequence (Gotschlich, et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); Murakami, et al., 20 Infect. Immun. 57:2318-2323 (1989)). Carbonetti et al. were the first to clone an entire gonococcal porin gene into E. coli using a tightly controlled pT7-5 expression plasmid. The results of these studies showed that when the porin gene was induced, very little porin protein accumulated and the expression of this protein was lethal to the E. coli (Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987)). In additional studies, Carbonetti et al. (Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)) did show that alterations in the gonococcal porin gene could be made in this system in E. coli and then reintroduced into gonococci. However, the ease with which one can make these manipulations and obtain enough porin 13protein for further biochemical and biophysical characterization seems limited.

Feavers et al. have described a method to amplify, by PCR, neisserial porin genes from a wide variety of sources using two synthesized oligonucleotides to common domains at the 5' and 3' ends of the porin genes respectively (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)). The oligonucleotides were constructed such that the amplified DNA could be forced cloned into plasmids using the restriction endonucleases BglII and Xhol.

Using the Feavers et al. PCR system, the DNA sequence of the mature PorB protein from meningococcal strain 8765 serotype 15 was amplified and ligated into the BamHI-XhoI site of the T7 expression plasmid pET-17b. This placed the mature PorB protein sequence in frame directly behind the T7 promoter and 20 amino acids of the 410 protein including the leader sequence. Upon addition of IPTG to a culture of E. coli containing this plasmid, large amounts of PorB protein accumulated within the bacteria. A complete explanation for why this construction was non-lethal to the E. coli and expressed large amount of the porin protein, await further studies. However, one possible hypothesis is that by 20 replacing the neisserial promoter and signal sequence with that of the T7 and 410 respectively, the porin product was directed to the cytoplasm rather than toward the outer membrane. Henning and co-workers have C.o reported that when E. coli OmpA protein and its fragments are expressed, those products which are found in the cytoplasm are less toxic than those 25 directed toward the periplasmic space (Klose, et al., J. Biol. Chem.

263:13291-13296 (1988); Klose, et al., J. Biol. Chem. 263:13297- 13302 (1988); Freudl, et al., J. Mol. Biol. 205:771-775 (1989)).

Whatever the explanation, once the PorB protein was expressed, it was easily isolated, purified and appeared to reform into trimers much like the 14native porin. The results of the inhibition ELISA data using human immune sera suggests that the PorB protein obtained in this fashion regains most if not all of the antigenic characteristics of the wild type PorB protein purified from meningococci. This expression system lends itself to the easy manipulation of the neisserial porin gene by modem molecular techniques.

In addition, this system allows one to obtain large quantities of pure porin protein for characterization. In addition, the present expression system allows the genes from numerous strains of Neisseria, both gonococci and meningococci, to be examined and characterized in a similar manner.

Thus, the present invention relates to a method of expressing an outer membrane meningococcal group B porin protein, in particular, the class 2 and class 3 porin proteins.

In one embodiment, the present invention relates to a method of expressing the outer membrane meningococcal group B porin protein in E. coli comprising: transforming E. coli by a vector comprising a selectable marker and a gene coding for a protein selected from the group consisting of: a mature porin protein, and (ii) a fusion protein comprising a mature porin protein fused to amino acids 1 to 20 or 22 of the T7 gene 010 capsid -protein; wherein said gene is operably linked to the T7 promoter; growing the transformed E. coli in a culture media containing a selection agent, and inducing expression of said protein; wherein the protein so produced comprises more than about 2% of the total protein expressed in the E. coli.

In a preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 5% of the total proteins expressed in E. coli. In another preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 10% of the total proteins expressed in E. coli. In yet another preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 30% of the total proteins expressed in E. coli.

Examples of plasmids which contain the T7 inducible promotor include the expression plasmids pET-17b, pET-11a, pET-24a-d(+) and pET-9a, all of which are commercially available from Novagen (565 Science Drive, Madison, WI 53711). These plasmids comprise, in sequence, a T7 promoter, optionally a lac operator, a ribosome binding site, restriction sites to allow insertion of the structural gene and a T7 terminator sequence. See, the Novagen catalogue, pages 36-43 (1993).

In a preferred embodiment, E. coli strain BL21 (DE3) AompA is employed. The above mentioned plasmids may be transformed into this strain or the wild-type strain BL21(DE3). E. coli strain BL21 (DE3) AompA is preferred as no OmpA protein is produced by this strain which 20 might contaminate the purified porin protein and create undesirable immunogenic side effects.

The transformed E. coli are grown in a medium containing a selection agent, e.g. any /3-lactam to which E. coli is sensitive such as ampicillin. The pET expression vectors provide selectable markers which 25 confer antibiotic resistance to the transformed organism.

High level expression of meningococcal group B porin protein can be toxic in E. coli. Surprisingly, the present invention allows E. coli to express the protein to a level of at least almost 30% and as high as of the total cellular proteins.

16 In another preferred embodiment, the present invention relates to a vaccine comprising the outer membrane meningococcal group B porin protein or fusion protein thereof, produced according to the abovedescribed methods, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the vaccine may be administered in an amount effective to elicit protective antibodies in an animal to Neisseria meningitidis. In a preferred embodiment, the animal is selected from the group consisting of humans, cattle, pigs, sheep, and chickens. In another preferred embodiment, the animal is a human.

In another preferred embodiment, the present invention relates to the above-described vaccine, wherein said outer membrane meningococcal group B porin protein or fusion protein thereof is conjugated to a meningococcal group B capsular polysaccharide Such capsular polysaccharides may be prepared as described in Ashton, F.E. et al., Microbial Pathog. 6:455-458 (1989); Jennings, H.J. et al., J. Immunol.

134:2651 (1985); Jennings, H.J. et al., J. Immunol. 137:1708-1713 (1986); Jennings, H.J. et al., J. Immunol. 142:3585-3591 (1989); Jennings, H.J., "Capsular Polysaccharides as Vaccine Candidates," in Current Topics in Microbiology and Immunology, 150:105-107 (1990); the contents of each o 20 of which are fully incorporated by reference herein.

Preferably, the CP is isolated according to Frasch, C.E., "Production and Control of Neisseria meningitidis Vaccines" in Bacterial Vaccines, Alan R. Liss, Inc., pages 123-145 (1990), the contents of which are fully incorporated by reference herein, as follows: Grow organisms in modified Franz medium 10 to 20 hrs 4 Heat kill, 55 0 C, 10 min Remove inactivated cells by centrifugation 4 Add Cetavlon to 0.1% Precipitate CP from culture broth 17- 4 Add calcium chloride to 1 M Dissolve CP then centrifuge to remove cellular debris 4 Add ethyl alcohol to Remove precipitated nucleic acids by centrifugation 4 Add ethyl alcohol to Precipitate crude CP and remove alcohol The crude CP is then further purified by gel filtration chromatography after partial depolymerization with dilute acid, e.g.

acetic acid, formic acid, and trifluoroacetic acid (0.01-0.5 to give a mixture of polysaccharides having an average molecular weight of 12,000- 16,000. The CP is then N-deacetylated with borohydride and Npropionylated to afford N-Pr GBMP. Thus, the CP that may be employed in the conjugate vaccines of the present invention may be CP fragments,

N-

deacylated CP and fragments thereof, as well as N-Pr CP and fragments thereof, so long as they induce active immunity when employed as part of a CP-porin protein conjugate (see the Examples).

In a further preferred embodiment, the present invention relates to a method of preparing a polysaccharide conjugate comprising: obtaining the above-described outer membrane meningococcal group B porin protein or fusion protein thereof; obtaining a CP from a Neisseria meningitidis organism; and conjugating the protein to the CP.

The conjugates of the invention may be formed by reacting the reducing end groups of the CP to primary amino groups of the porin by reductive amination. The reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both.

Preferably, the CP is conjugated to the porin protein by the method of Jennings et al., U.S. Patent No. 4,356,170, the contents of which are fully o.oe, 18 incorporated by reference herein, which involves controlled oxidation of the CP with periodate followed by reductive amination with the porin protein.

The vaccine of the present invention comprises the meningococcal group B porin protein, fusion protein or conjugate vaccine in an amount effective depending on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, the meningococcal group B porin protein, fusion protein or vaccine of the present invention can also be administered by an intraperitoneal or intravenous route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts might be expected to fall within the range of 2 micrograms of the protein per kg body weight to 100 micrograms per kg body weight.

The vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the meningococcal group B porin protein, fusion protein or conjugate vaccine have suitable solubility properties. The vaccines may 20 be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al.

University Park Press, Baltimore, MD (1978), for methods of 25 preparing and using vaccines.

The meningococcal group B porin protein, fusion protein or conjugate vaccines of the present invention may further comprise adjuvants which enhance production of porin-specific antibodies. Such adjuvants include, but are not limited to, various oil formulations such as Freund's 19- 20 .9 *oo complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Patent No.

4,258,029), the dipeptide known as MDP, saponin, aluminum hydroxide, and lymphatic cytokine.

Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) or ST may be used for administration to a human. The meningococcal group B porin protein or a conjugate vaccine thereof may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The meningococcal group B porin protein or conjugate vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Patent No. 4,235,877.

In another preferred embodiment, the present invention relates to a method of preventing bacterial meningitis in an animal comprising administering to the animal the meningococcal group B porin protein, fusion protein or conjugate vaccine produced according to methods described in an amount effective to prevent bacterial meningitis.

In a further embodiment, the invention relates to a method of purifying the above-described outer membrane meningococcal group

B

porin protein or fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein by gel filtration.

The lysing step may be carried out according to any method known to those of ordinary skill in the art, e.g. by sonication, enzyme digestion, osmotic shock, or by passing through a mull press.

9 9 The inclusion bodies may be washed with any buffer which is capable of solubilizing the E. coli cellular proteins without solubilizing the inclusion bodies comprising the meningococcal group B porin protein.

Such buffers include but are not limited to TEN buffer (50 mM Tris HC1, 1 mM EDTA, 100 mM NaCI, pH Tricine, Bicine and HEPES.

Denaturants which may be used in the practice of the invention include 2 to 8 M urea or about 2 to 6 M guanidine HC1, more preferably, 4 to 8 M urea or about 4 to 6 M guanidine HC1, and most preferably, about 8 M urea or about 6 M guanidine

HCI).

Examples of detergents which can be used to dilute the solubilized meningococcal group B porin protein include, but are not limited to, ionic detergents such as SDS and cetavlon (Calbiochem); non-ionic detergents such as Tween, Triton X, Brij 35 and octyl glucoside; and zwitterionic detergents such as 3 ,1 4 -Zwittergent, empigen BB and Champs.

Finally, the solubilized outer membrane meningococcal group

B

porin protein may be purified by gel filtration to separate the high and low molecular weight materials. Types of filtration gels include but are not limited to Sephacryl-300, Sepharose CL-6B, and Bio-Gel A-1.5m. The column is eluted with the buffer used to dilute the solubilized protein. The fractions containing the porin or fusion thereof may then be identified by :.gel electrophoresis, the fractions pooled, dialyzed, and concentrated.

Finally, substantially pure 95%) porin protein and fusion protein may be obtained by passing the concentrated fractions through a Q sepharose high performance column.

o o 25 In another embodiment, the present invention relates to expression of the meningococcal group B porin protein gene which is part of a vector Swhich comprises the T7 promoter, which is inducible. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. The T7 promoter is inducible by the addition of a too o -21 isopropyl f-D-thiogalactopyranoside (IPTG) to the culture medium.

Alternatively, the Tac promotor or heat shock promotor may be employed.

Preferably, the meningococcal group B porin protein gene is expressed from the pET-17 expression vector or the pET-lla expression vector, both of which contain the T7 promoter.

The cloning of the meningococcal group B porin protein gene or fusion gene into an expression vector may be carried out in accordance with conventional techniques, including blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Reference is made to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989), for general methods of cloning.

The meningococcal group B porin protein and fusion protein expressed according to the present invention must be properly refolded in order to achieve a structure which is immunologically characteristic of the native protein. In yet another embodiment, the present invention relates to a method of refolding the above-described outer membrane protein and 20 fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the 25 resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein or fusion protein by gel filtration to give the refolded protein in the eluant. Surprisingly, it has been discovered that the folded trimeric meningococcal group B class 2 and class 3 porin -22proteins and fusion proteins are obtained directly in the eluant from the gel filtration column.

In another preferred embodiment, the present invention relates to a substantially pure refolded outer membrane meningococcal group B porin protein and fusion protein produced according to the above-described methods. A substantially pure protein is a protein that is generally lacking in other cellular Neisseria meningitidis components as evidenced by, for example, electrophoresis. Such substantially pure proteins have a purity of as measured by densitometry on an electrophoretic gel after staining with Coomassie blue or silver stains.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in this art which are obvious to those skilled in the art are within the spirit and scope of the present invention.

Examples

S

Example 1. Cloning of the Class 3 Porin Protein from Group B Neisseria meningitidis see**:

S.

S Materials and Methods 20 Organisms: The Group B Neisseria meningitidis strain 8765 (B:15:P1,3) was obtained from Dr. Wendell Zollinger (Walter Reed Army Institute for Research) and grown on agar media previously described (Swanson, Infect. Immun. 21:292-302 (1978)) in a candle extinction jar in an incubator maintained at 30°C. Escherichia coli strains DME558 25 (from the collection of S. Benson; Silhavy, T.J. et al., "Experiments with Gene Fusions," Cold Spring Harbor Laboratory, Cold Spring Harbor, -23- 1984), BRE51 (Bremer, E. etal., FEMSMicrobiol. Lett. 33:173-178 (1986)) and BL21(DE3) were grown on LB agar plates at 37°C.

PI Transduction: A PI,, lysate of E. coli strain DME558 was used to transduce a tetracycline resistance marker to strain BRE51 (Bremer,

E.,

et al., FEMSMicrobiol Lett. 33:173-178 (1986)) in which the entire ompA gene had been deleted (Silhavy, et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984)).

Strain DME558, containing the tetracycline resistance marker in close proximity of the ompA gene, was grown in LB medium until it reached a density of approximately 0.6 OD at 600 nm. One tenth of a milliliter of M CaCI, was added to the 10 ml culture and 0.1 ml of a solution containing 1 x 10 9 PFU of Plvir. The culture was incubated for 3 hours at 37 0 C. After this time, the bacterial cell density was visibly reduced.

ml of chloroform was added and the phage culture stored at 4°C.

Because typically 1-2% of the E. coli chromosome can be packaged in each phage, the number of phage generated covers the entire bacterial host chromosome, including the tetracycline resistance marker close to the ompA gene.

SNext, strain BRE51, which lacks the ompA gene, was grown in LB 20 medium overnight at 37 0 C. The overnight culture was diluted 1:50 into fresh LB and grown for 2 hr. The cells were removed by centrifugation and resuspended in MC salts. 0.1 ml of the bacterial cells were mixed with .0.05 of the phage lysate described above and incubated for 20 min. at room temperature. Thereafter, an equal volume of 1 M sodium citrate was added and the bacterial cells were plated out onto LB plates containing 12.5 /g/ml Sof tetracycline. The plates were incubated overnight at 370C.

Tetracycline resistant (12 /g/ml) transductants were screened for lack of OmpA protein expression by SDS-PAGE and Western Blot analysis, as described below. The bacteria resistant to the antibiotic have the -24tetracycline resistance gene integrated into the chromosome very near where the ompA gene had been deleted from this strain. One particular strain was designated BRE-TR.

A second round of phage production was then carried out with the strain BRE-TR, using the same method as described above. Representatives of this phage population contain both the tetracycline resistance gene and the OmpA deletion. These phage were then collected and stored. These phage were then used to infect E. coli BL21(DE3). After infection, the bacteria contain the tetracycline resistance marker. In addition, there is a high probability that the OmpA deletion was selected on the LB plates containing tetracycline.

Colonies of bacteria which grew on the plates were grown up separately in LB medium and tested for the presence of the OmpA protein.

Of those colonies selected for examination, all lacked the OmpA protein as judged by antibody reactivity on SDS-PAGE western blots.

SDS-PAGE and Western Blot: The SDS-PAGE was a variation of Laemmli's method (Laemmli, Nature 227:680-685 (1970)) as described previously (Blake and Gotschlich, J. Exp. Med. 159:452-462 (1984)). Electrophoretic transfer to Immobilon P (Millipore Corp. Bedford, 20 MA) was performed according to the methods of Towbin et al.. (Towbin, et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979)) with the exception that the paper was first wetted in methanol. The Western blots were probed with phosphatase conjugated reagents (Blake, et al., Analyt. Biochem. 136:175-179 (1984)).

Polymerase Chain Reaction: The method described by Feavers et al. (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)) was used Sto amplify the gene encoding the PorB. The primers selected were primers 33 (SEQ ID NO. 11) (GGG GTA GAT CTG CAG GTT ACC TTG TAC GGT ACA ATT AAA GCA GGC GT) and 34 (SEQ ID NO. 12) (GGG GGG GTG ACC CTC GAG TTA GAA TTT GTG ACG CAG ACC AAC) as previously described (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)). Briefly, the reaction components were as follows: Meningococcal strain 8765 chromosomal DNA (100 1 5' and 3' primers (1 pM) 2 pl each; dNTP (10 mM stocks), 4 pl each; 10 X PCR reaction buffer (100 mM Tris HC1, 500 mM KC1, pH 10 p1; 25 mM MgC1 2 6 pl; double distilled Hz0, 62 pl; and Taq polymerase (Cetus Corp., 5 u/pl), 1 pA. The reaction was carried out in a GTC-2 Genetic Thermocycler (Precision Inst. Inc Chicago, IL) connected to a Lauda 4/K methanol/water cooling system (Brin n Istruments, Inc., Westbury, NY) set at 0°C.

The thermocycler was programmed to cycle 30 times through: 94°C, 2 min.; 40 0 C, 2 min.; and 72°C, 3 min. At the end of these 30 cycles, the reaction was extended at 72°C for 3 min and finally held at 4 0 C until readied for analysis on a 1% agarose gel in TAE buffer as described by Maniatis (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).

Subcloning of the PCR product: The pET-17b plasmid (Novagen, Inc.) was used for subcloning and was prepared by double digesting the plasmid with the restriction endonucleases BamHI and XhoI (New England 20 Biolabs, Inc., Beverly, MA). The digested ends were then dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN). The digested plasmid was then analyzed on a 1% agarose gel, the cut plasmid removed, and purified using the GeneClean kit (Biol01, La Jolla, CA). The PCR product was prepared by 25 extraction with phenol-chloroform, chloroform, and finally purified using the GeneClean Kit (BiolOl). The PCR product was digested with restriction endonucleases Bglll and XhoI (New England Biolabs, Inc.).

The DNA was then extracted with phenol-chloroform, precipitated by adding 0.1 volumes of 3 M sodium acetate, 5 p1 glycogen (20 pg/l), and -26volumes of ethanol. After washing the DNA with 70% ethanol (vol/vol), it was redissolved in TE buffer. The digested PCR product was ligated to the double digested pET-17b plasmid described above using the standard T4 ligase procedure at 16"C overnight (Current Protocols in Molecular Biology, John Wiley Sons, New York (1993)). The ligation product was then transformed into the BL21 (DE3)-AompA described above which were made competent by the method of Chung et al. (Chung, C.T., etal., Proc. Natl. Acad. Sci. USA 86:2172-2175 (1989)). The transformants were selected on LB plates containing 50 pjg/ml carbenicillin and 12gg/ml tetracycline. Several transformants were selected, cultured in LB both containing carbenicillin and tetracycline for 6 hours at 30 0 C, and plasmid gene expression inducted by the addition of IPTG. The temperature was raised to 37°C and the cultures continued for an additional 2 hrs. The cells of each culture were collected by centrifugation, whole cell lysates prepared, and analyzed by SDS-PAGE and Western Blot using a monoclonal antibody (4D11) which reacts with all neisserial porins.

Nucleotide Sequence Analysis: The nucleotide sequences of the cloned Class 3 porin gene DNA were determined by the dideoxy method S• using denatured double-stranded plasmid DNA as the template as described 20 (Current Protocols in Molecular Biology, John Wiley Sons, New York (1993)). Sequenase II kits (United States Biochemical Corp., Cleveland, OH) were used in accordance with the manufacturer's instructions. The S* three synthesized oligonucleotide primers (Operon Technologies, Inc., Alameda, CA) were used for these reactions. One for the 5' end(SEQ ID 25 NO. 13) which consisted of5'TCAAGCTTGGTACCGAGCTCand two for the 3' end, (SEQ ID NO. 14) 5'TTTGTTAGCAGCCGGATCTG (SEQ 1D NO. 15) and 5' CTCAAGACCCGTTTAGAGGCC. Overlapping, nested deletions were made by linearizing the plasmid DNA by restriction endonuclease Bpul 1021 and the ends blunted by the addition of Thio-dNTP -27and Klenow polymerase (Current Protocols in Molecular Biology, John Wiley Sons, New York (1993)). The linearized plasmid was then cleaved with restriction endonuclease Xhol and the exoII/Mung bean nuclease deletion kit used to make 3' deletions of the plasmid (Stratagene, Inc., La Jolla, CA) as instructed by the supplier. A map of this strategy is shown in Figure 1.

Expression and purification of the PorB gene product: Using a sterile micropipette tip, a single colony of the BL21 (DE3)-AopA containing the PorB-pET-17b plasmid was selected and inoculated into ml of LB broth containing. 50 /g/ml carbenicillin. The culture was incubated overnight at 30 0 C while shaking. The 10 ml overnight culture was then sterily added to 1 liter of LB broth with the same concentration of carbenicillin, and the culture continued in a shaking incubator at 37°C until the OD6 reached 0.6-1.0. Three mis of a stock solution of IPTG (100 mM) was added to the culture and the culture incubated for an additional 30 min. Rifampicin was then added (5.88 ml of a stock solution; 34 mg/ml in methanol) and the culture continued for an additional 2 hrs.

SThe cells were harvested by centrifugation at 10,000 rpm in a GS3 rotor for 10 min and weighed. The cells were thoroughly resuspended in 3 ml 20 of TEN buffer (50 mM Tris HC1, 1 mM Tris HCI, 1 mM EDTA, 100 mM Na C 1 pH 8.0) per gram wet weight of cells. To this was added 8 l of PMSF stock solution (50 mM in anhydrous ethanol) and 80 ul of a lysozyme stock solution (10 mg/ml in water) per gram wet weight of cells.

This mixture was stirred at room temperature for 20 min. While stirring, 4 mg per gram wet weight of cells of deoxycholate was added. The mixture was placed in a 37 0 C water bath and stirred with a glass rod.

When the mixture became viscous, 20 /l of DNase I stock solution (1 mg/ml) was added per gram weight wet cells. The mixture was then removed from the water bath and left at room temperature until the solution -28was no longer viscous. The mixture was then centrifuged at 15,000 rpm in a SS-34 rotor for 20 min at 4 C. The pellet was retained and thoroughly washed twice with TEN buffer. The pellet was then resuspended in freshly prepared TEN buffer containing 0.1 mM PMSF and 8 M urea and sonicated in a bath sonicator (Heat Systems, Inc., Plainview, NY). The protein concentration was determined using a BCA kit (Pierce, Rockville, IL) and the protein concentration adjusted to less than 10 mg/ml using the TEN-urea buffer. The sample was then diluted 1:1 with 10% (weight/vol) Zwittergent 3,14 (CalBiochem, La Jolla, CA), sonicated, and loaded onto a Sephacryl S-300 molecular sieve column. The Sephacryl S-300 column cm x 200 cm) had previously equilibrated with 100 mM Tris HC1, 200 mM NaCI, 10 mM EDTA, 0.05% Zwittergent 3,14, and 0.02% azide, pH The column flow rate was adjusted to 8 ml/hr and 10 ml fractions were collected. The OD 2 o of each fraction was measured and SDS-PAGE analysis performed on protein containing fractions.

Inhibition ELISA Assays: Microtiter plates (Nunc-Immuno Plate IIF, Nun c Inc., Naperville, IL) were sensitized by adding 0.1 ml per well of porB (2 Itg/ml) purified from the wild type strain 8765, in 0.1 M carbonate buffer, pH 9.6 with 0.02% azide. The plates were incubated 20 overnight at room temperature. The plates were washed five times with 0.9% NaC1, 0.05% Brij 35, 10 mM sodium acetate pH 7.0, 0.02% azide.

Human immune sera raised against the Type 15 Class 3 PorB protein was obtained from Dr. Phillip O. Livingston, Memorial-Sloan Kettering Cancer Center, New York, N.Y. The human immune sera was diluted in PBS with 0.5% Brij 35 and added to the plate and incubated for 2 hr at room temperature. The plates were again washed as before and the secondary antibody, alkaline phosphatase conjugated goat anti-human IgG (Tago Inc., Burlingame, CA), was diluted in PBS-Brij, added to the plates and incubated for 1 hr at room temperature. The plates were washed as before -29- *0*a 20 a a **r and p-nitrophenyl phosphate (Sigma Phosphatase Substrate 104) (1 mg/ml) in 0.1 diethanolamine, 1 mM MgCI 2 0.1 mM ZnCI, 0.02% azide, pH 9.8, was added. The plates were incubated at 37 0 C for 1 h and the absorbance at 405 nm determined using an Elida-5 microtiter plate reader (Physica, New York, NY). Control wells lacked either the primary and/or secondary antibody. This was done to obtain a titer for each human serum which would give a half-maximal reading in the ELISA assay. This titer for each human serum would be used in the inhibition ELISA. The ELISA microtiter plate would be sensitized with purified wild type PorB protein and washed as before. In a separate V-96 polypropylene microtiter plate (Nunc, Inc.), varying amounts of either purified wild type PorB protein or the purified recombinant PorB protein were added in a total volume of pl. The human sera were diluted in PBS-Brij solution to twice their half maximal titer and 75 l1 added to each of the wells containing the PorB or recombinant PorB proteins. This plate was incubated for 2 hr at room temperature and centrifuged in a Sorvall RT6000 refrigerated centrifuge, equipped with microtiter plate carriers (Wilmington, DE) at 3000 rpm for 10 min. Avoiding the V-bottom, 100 p from each well was removed and transferred to the sensitized and washed ELISA microtiter plate. The ELISA plates are incubated for an additional 2 hr, washed, and the conjugated second antibody added as before. The plate is then processed and read as described. The percentage of inhibition is then processed and read as described. The percentage of inhibition is calculated as follows: 1 (ELISA value with either PorB or rPorB protein added) (ELISA value without the porB added) Results Polymerase Chain Reaction and Subcloning: A method to easily clone, genetically manipulate, and eventually obtain enough pure porin protein from any number of different neisserial porin genes for further antigenic and biophysical characterization has been developed. The first step toward this goal was cloning the porin gene from a Neisseria. Using a technique originally described by Feavers, et al. (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)), the DNA sequence of the mature porin protein from a class 3, serotype 15 porin was amplified using the chromosome of meningococcal strain 8765 as a template for the PCR reaction. Appropriate endonuclease restriction sites had been synthesized onto the ends of the oligonucleotide primers, such that when cleaved, the amplified mature porin sequence could be directly ligated and cloned into the chosen expression plasmid. After 30 cycles, the PCR products shown in Figure 2 were obtained. The major product migrated between 900bp and 1000bp which was in accord with the previous study (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)). However, a higher molecular weight product was not seen, even though the PCR was conducted under low annealing stringencies (40 0 C; 50 mM KCI).

To be able to produce large amounts of the cloned porin protein, the tightly controlled expression system of Studier, et al. (Studier and Moffatt, J. Mol. Biol. 189:113-130 (1986)) was employed, which is commercially available through Novagen Inc. The amplified PCR product was cloned into the BamHI-Xhol site of plasmid pET-17b. This strategy places the S 25 DNA sequence for the mature porin protein in frame directly behind the T7 promoter, the DNA sequence encoding for the 9 amino acid leader sequence and 11 amino acids of the mature 410 protein. The Studier E.

coli strain BL21 lysogenic for the DE3 lambda derivative (Studier and -31 Moffatt, J. Mol. Biol. 189:113-130 (1986)) was selected as the expression host for the pET-17b plasmid containing the porin gene. But because it was thought that the OmpA protein, originating from the E. coli expression host, might tend to co-purify with the expressed meningococcal porin protein, a modification of this strain was made by P1 transduction which eliminated the ompA gene from this strain. Thus, after restriction endonuclease digestion of both the PCR product and the pET-17b vector and ligation, the product was transformed into BL21(DE3)-AompA and transformants selected for ampicillin and tetracycline resistance. Of the numerous colonies observed on the selection plate, 10 were picked for further characterization. All ten expressed large amounts of a protein, which migrated at the approximate molecular weight of the PorB protein, when grown to log phase and induced with IPTG. The whole cell lysate of one such culture is shown in Figure 3a. The western blot analysis with the 4D11 monoclonal antibody further suggested that the protein being expressed was the PorB protein (Figure 3b). As opposed to other studies, when neisserial porins have been cloned and expressed in E. coli, the host bacterial cells showed no signs of any toxic or lethal effects even after the addition of the IPTG. The E. coli cells appeared viable and could be 20 recultured at any time throughout the expression phase.

Nucleotide sequence analysis: The amount of PorB expressed in these experiments was significantly greater than that previously observed and there appeared to be no adverse effects of this expression on the host E. coli. To be certain that no PCR artifacts had been introduced into the 25 meningococcal porin gene to allow for such high expression, the entire 010 porin fusion was sequenced by double stranded primer extension from the plasmid. The results are shown in Figure 4. The nucleotide sequence was identical with another meningococcal serotype 15 PorB gene sequence previously reported by Heckels, et al. (Ward, et al., FEMS 32- Microbiol. Lett. 73:283-289 (1992)) with two exceptions which are shown.

These two nucleotide differences each occur in the third position of the codon and would not alter the amino acid sequence of the expressed protein. Thus, from the nucleotide sequence, there did not appear to be any PCR artifact or mutation which might account for the high protein expression and lack of toxicity within the E. coli. Furthermore, this data would suggest that a true PorB protein was being produced.

Purification of the expressed porB gene product: The PorB protein expressed in the E. coli was insoluble in TEN buffer which suggested that when expressed, the PorB protein formed into inclusion bodies. However, washing of the insoluble PorB protein with TEN buffer removed most of the contaminating E. coli proteins. The PorB protein could then be solubilized in freshly prepared 8M urea and diluted into the Zwittergent 3,14 detergent. The final purification was accomplished, using a Sephacryl S-300 molecular sieve column which not only removed the urea but also the remaining contaminating proteins. The majority of the PorB protein eluted from the column having the apparent molecular weight of trimers much like the wild type PorB. The comparative elution patterns of both the wild type and the PorB expressed in the E. coli are shown in Figure 5. It is 20 important to note that when the PorB protein concentration in the 8 M urea was in excess of 10 mg/ml prior to dilution into the Zwittergent detergent, the relative amounts of PorB protein found as trimers decreased and appeared as aggregates eluting at the void volume. However,. at protein concentrations below 10 mg/ml in the urea buffer, the majority of the PorB a S 25 eluted in the exact same fraction as did the wild type PorB. It was also determined using a T7-Tag monoclonal antibody and western blot analysis that the 11 amino acids of the mature T7 capsid protein were retained as the amino terminus. The total yield of the meningococcal porin protein from one liter of E. coli was approximately 50 mg.

-33- Inhibition ELISA Assays. In order to determine if the purified trimeric recombinant PorB had a similar antigenic conformation as compared to the PorB produced in the wild type meningococcal strain 8765, the sera from six patients which had been vaccinated with the wild type meningococcal Type 15 PorB protein were used in inhibition

ELISA

assays. In the inhibition assay, antibodies reactive to the native PorB were competitively inhibited with various amounts of either the purified recombinant PorB or the homologous purified wild type PorB. The results of the inhibition with the homologous purified PorB of each of the six human sera and the mean inhibition of these sera.are shown in Figure 6.

The corresponding inhibition of these sera with the purified recombinant PorB is seen in Figure 6b. A comparison of the mean inhibition from Figure 6 and 7 are plotted in Figure 8. These data would suggest that the antibodies contained in the sera of these six patients found similar epitopes on both the homologous purified wild- type PorB and the purified recombinant PorB. This gave further evidence that the recombinant PorB had regained most if not all of the native conformation found in the wild type PorB.

0 Example 2. Cloning of the Class 2 Porin from Group B Neisseria Meningitidis strain BNCV M986 Genomic DNA was isolated from approximately 0.5g of Group B Neisseria meningitidis strain BNCV M986 (serotype 2a) using previously described methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor 25 Laboratory Press (1989)). This DNA then served as the template for two class 2 porin specific oligonucleotides in a standard PCR reaction. These oligonucleotides were designed to be complementary to the 5' and 3' flanking regions of the class 2 porin and to contain EcoRI restriction sites -34to facilitate the cloning of the fragment. The sequences of the oligonucleotides were as follows: (SEQ ID NO. 16) AGC GGC TTG GAA TTC CCG GCT GGC TTA AAT TTC 3' (SEQ ID NO. 17) and 5' CAA ACG AAT GAA TTC AAA TAA AAA AGC CTG 3'.

The polymerase chain reaction was then utilized to obtain the class 2 porin.

The reaction conditions were as follows: BNCV M986 genomic DNA 200ng, the two oligonucleotide primers described above at 1 gM of each, 200 gM of each dNTP, PCR reaction buffer (10 mM Tris HCI, 50 mM KC1, pH 1.5 mM MgCI 2 and 2.5 units of Taq polymerase, made up to 100 1 with distilled H20. This reaction mixture was then subjected to cycles of 95°C for 1 min, 50 0 C for 2 min and 72°C for 1.5 min. At the end of the cycling period, the reaction mixture was loaded on a 1% agarose gel and the material was electrophoresed for 2h after which the band at 1.3 kb was removed and the DNA recovered using the Gene Clean kit (Bio 101). This DNA was then digested with EcoRI, repurified and S...re ligated to EcoRI digested pUC19 using T 4 DNA ligase. The ligation mixture was used to transform competent E. coli DH5ca. Recombinant plasmids were selected and sequenced. The insert was found to have a 20 DNA sequence consistent with that of a class 2 porin. See, Murakami,

K.

et al., Infect. Immun. 57:2318-2323 (1989).

The plasmid pET-17b (Novagen) was used to express the class 2 porin. As described below, two plasmids were constructed that yielded two different proteins. One plasmid was designed to produce a mature class 2 porin while the other was designed to yield a class 2 porin fused to .:amino acids from the T7 gene 0410 capsid protein.

Construction of the mature class 2 porin The mature class 2 porin was constructed by amplifying the pUC19class 2 porin construct using the oligonucleotides (SEQ ID NO. 18) 5' CCT GTT GCA GCA CAT ATG GAC GTT ACC TTG TAC GGT ACA ATT AAA GC 3' and (SEQ ID NO. 19) 5 CGA CAG GCT TTT TCT CGA GAC CAA TCT TTT CAG This strategy allowed the cloning of the amplified class 2 porin into the Ndel and Xhol sites of the plasmid pET-17b thus producing a mature class 2 porin. Standard PCR was conducted using the pUC19-class 2 as the template and the two oligonucleotides described above. This PCR reaction yielded a 1.1kb product when analyzed on a agarose gel. The DNA obtained from the PCR reaction was gel purified and digested with the restriction enzymes Ndel and Xhol. The 1.lkb DNA produced was again gel purified and ligated to Ndel and Xhol digested pET-17b using T 4 DNA ligase. This ligation mixture was then used to transform competent E. coli DH5a. Colonies that contained the 1.1kb insert were chosen for further analysis. The DNA from the clones was analyzed by restriction mapping and the cloning junctions of the chosen plasmids were sequenced. After this analysis, the DNA obtained from the D H 5 ca clones was used to transform E. coli BL21(DE3)-AompA.

The transformants were selected to LB-agar containing 100 /Pg/ml of carbenicillin. Several transformants were screened for their ability to make the class 2 porin protein. This was done by growing the clones in LB :...liquid medium containing 100 pg/ml of carbenicillin and 0.4% glucose at 30 0 C to ODo 0.6 then inducing the cultures with IPTG (0.4 mM). The cells were then disrupted and the cell extract was analyzed by SDS-PAGE.

-36- Construction of the fusion class 2 porin The fusion class 2 porin was constructed by amplifying the pUC19class 2 porin construct using the oligonucleotides (SEQ ID NO. 20) 5' CCT GTT GCA GCG GAT CCA GAC GTT ACC TTG TAC GGT ACA ATT AAA GC 3' and (SEQ ID NO. 21) 5' CGA CAG GCT TTT TCT CGA GAC CAA TCT TTT CAG This strategy allowed the cloning of the amplified class 2 porin into the BamHI and XhoI sites of the plasmid pET- 17b thus producing a fusion class 2 porin containing an additional 22 amino acids at the N-terminus derived from the T7 410 capsid protein contained in the plasmid. Standard PCR was conducted using the pUC19-class 2 as the template and the two oligonucleotides described above. The PCR reaction yielded a 1.1kb product when analyzed on a 1.0% agarose gel.

The DNA obtained from the PCR reaction was gel purified and digested with the reaction enzymes BamHI and XhoI. The 1. kb product produced was again gel purified and ligated to BamHI and XhoI digested pET-17b using T 4 DNA ligase. This ligation mixture was then used to transform competent E. coli DH5a. Colonies that contained the 1.1kb insert were chosen for further analysis. The DNA from the DH5a clones was analyzed by restriction enzyme mapping and the cloning junctions of the chosen plasmids were sequenced. After this analysis, the DNA obtained from the DH5a clones was used to transform E. coli BL21(DE3)-AompA. The transformants were selected on LB-agar containing 100 .g/ml of carbenicillin. Several transformants were screened for their ability to make the class 2 porin protein. This was done by growing the clones in LB S 25 liquid medium containing 100 pg/ml of carbenicillin and 0.4% glucose at 30 0C to ODwo 0.6 then inducing the cultures with IPTG (0.4 mM). The cells were then disrupted and the cell extract was analyzed by SDS-PAGE.

-37- Example 3. Cloning and Expression of the Mature class 3 porin from Group B Neisseria meningitidis strain 8765 in E. coli Genomic DNA was isolated from approximately 0.5 g of Group B Neisseria meningitidis strain 8765 using the method described above (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989)). This DNA then served as the template for two class 3 porin specific oligonucleotides in a standard PCR reaction.

The mature class 3 porin was constructed by amplifying the genomic DNA from 8765 using the oligonucleotides: (SEQ ID NO. 22) 5' GTT GCA GCA CATATG GAC GTT ACC CTG TAC GGC ACC 3' (SEQ ID NO. 23) and 5' GGG GGG ATG GAT CCA GAT TAG AAT TTG TGG CGC AGA CCG ACA CC This strategy allowed the cloning of the amplified class 3 porin into the Ndel and BamH sites of the plasmid pETthus producing a mature class 3 porin. Standard PCR was conducted using the genomic DNA isolated from 8765 as the template and the two oligonucleotides described above..

The reaction conditions were as follows: 8765 genomic DNA 200 ng, the two oligonucleotide primers described above at 1 pM of each, 200 20 AM of each dNTP, PCR reaction buffer (10 mM Tris HC1, 50 mM KC1, pH 1.5 mM MgCl2, and 2.5 units of Taq polymerase, and made up to 100 Al with distilled water. This reaction mixture was then subjected to cycles of 95°C for 1 min, 50°C for 2 min and 72°C for 1.5 min.

This PCR reaction yielded about 930 bp of product, as analyzed on 25 a 1% agarose gel. The DNA obtained from the PCR reaction was gel purified and digested with the restriction enzymes NdeI and BamHI. The 930 bp product was again gel purified and ligated to NdeI and BamHI digested pET-24a(+) using T4 ligase. This ligation mixture was then used to transform competent E. coli DH5. Colonies that contained the 930 bp -38 insert were chosen for further analysis. The DNA from the E. coli clones was analyzed by restriction enzyme mapping and cloning junctions of the chosen plasmids were sequenced. After this analysis, the DNA obtained from the E. coli DH5a clones was used to transform E. coli BL21(DE3)-AompA. The transformants were selected on LB-agar containing 50 pg/ml of kanamycin. Several transformants were screened for their ability to make the class 3 porin protein. This was done by growing the clones in LB liquid medium containing 50 pg/ml of kanamycin and 0.4% of glucose at 30 0 C to OD0 0.6 then inducing the cultures with IPTG (1 mM). The cells were then disrupted and the cell extract was analyzed by SDS-PAGE.

Example 4. Purification and refolding of recombinant class 2 porin E coli strain BL21(DE3)AompA [pNV-5] is grown to mid-log phase (OD 0.6 at 600 nm) in Luria broth at 30 0 C. IPTG is then added (0.4 mM final) and the cells grown an additional two hours at 37 0 C. The cells were then harvested and washed with several volumes of TEN buffer mM Tris-HCI, 0.2 M NaCI, 10 mM EDTA, pH 8.0) and the cell paste stored frozen at -75 C.

For purification preweighed cells are thawed and suspended in TEN 20 buffer at a 1:15 ratio The suspension is passed through a Stansted cell disrupter (Stansted fluid power Ltd.) twice at 8,000 psi. The resultant solution is then centrifuged at 13,000 rpm for 20 min and the supernatant discarded. The pellet is then twice suspended in TEN buffer containing 0.5% deoxycholate and the supernatants discarded. The pellet is then 25 suspended in TEN buffer containing 8 M deionized urea (electrophoresis grade) and 0.1 mM PMSF (3 g/lOml). The suspension is sonicated for min or until an even suspension is achieved. 10 ml of a 10% aqueous -39solution of 3,14-zwittergen (Calbiochem) is added and the solution thoroughly mixed. The solution is again sonicated for 10 min. Any residual insoluble material is removed by centrifugation. The protein concentration is determined and the protein concentration adjusted to 2 mg/ml with 8 M urea-10% zwittergen buffer (1:1 ratio).

This mixture is then applied to a 2.6 x 100 cm column of Sephacryl S-300 equilibrated in 100 mM Tris-HC1, 1 M NaCI, 10 mM EDTA, mM CaC 2 0.05% 3 ,14-zwittergen, 0.02% sodium azide, pH 8.0. The flow rate is maintained at 1 ml/min. Fractions of 10 ml are collected. The porin refolds into trimer during the gel filtration. The OD 280 nm of each fraction is measured and those fractions containing protein are subjected to SDS gel electrophoresis assay for porin. Those fractions containing porin are pooled. The pooled fractions are either dialyzed or diluted 1:10 in 50 mM Tris HC1pH 8.0, 0.05% 3 ,14-zwittergen, 5 mM EDTA, 0.1 M NaCI. The resulting solution is then applied to a 2.6 x cm Q sepharose high performance column (Pharmacia) equilibrated in the same buffer. The porin is eluted with a linear gradient of 0.1 to 1 M NaCI.

o Example 5. Purification and refolding of recombinant class 3 porin 20 E coli strain BL21 (DE3) AompA containing the porB-pET-17b plasmid is grown to mid-log phase (OD 0.6 at 600 nm) in Luria broth at 30 0 C. IPTG is then added (0.4 mM final) and the cells grown an additional two hours at 37 0 C. The cells were then harvested and washed with several volumes of TEN buffer (50 mM Tris-HC1, 0.2 M NaCI, 25 mM EDTA, pH 8.0) and the cell paste stored frozen at For purification about 3 grams of cells are thawed and suspended in 9 ml of TEN buffer. Lysozyme is added (Sigma, 0.25 mg/ml) 40 deoxycholate (Sigma, 1.3 mg/ml) plus PMSF (Sigma, /g/ml) and the mixture gently shaken for one hour at room temperature. During this time, the cells lyse and the released DNA causes the solution to become very viscous. DNase is then added (Sigma, 2 yg/ml) and the solution again mixed for one hour at room temperature. The mixture is then centrifuged at 15K rpm in a S-600 rotor for 30 minutes and the supernatant discarded.

The pellet is then twice suspended in 10 ml of TEN buffer and the supernatants discarded. The pellet is then suspended in 10 ml of 8 M urea (Pierce) in TEN buffer. The mixture is gently stirred to break up any clumps. The suspension is sonicated for 20 minutes or until an even suspension is achieved. 10 ml of a 10% aqueous solution of 3,14zwittergen (Calbiochem) is added and the solution thoroughly mixed. The solution is again sonicated for 10 minutes. Any residual insoluble material is removed by centrifugation. The protein concentration is determined and the protein concentration adjusted to 2 mg/ml with 8 M zwittergen buffer (1:1 ratio).

"This mixture is then applied to a 180 x 2.5 cm column of Sephacryl S-300 (Pharmacia) equilibrated in 100 mM Tris-HCI, 1 M NaCI, 10 mM EDTA, 20 mM CaCI 2 0.05% 3,14-zwittergen, pH 8.0. The flow rate 20 is maintained at 1 ml/min. Fractions of 10 ml are collected. The porin refolds into trimer during the gel filtration. The ODuo nm of each fraction is measured and those fractions containing protein are subjected to SDS gel electrophoresis assay for porin. Those fractions containing porin are pooled.

S 25 The pooled fractions are dialyzed and concentrated 4-6 fold using Amicon concentrator with a PM 10 membrane against buffer containing 100 mM Tris-HC1, 0.1 M NaCI, 10 mM EDTA, 0.05% 3,14-zwittergen, pH 8.0. Alternatively, the pooled fractions are precipitated with ethanol and resuspended with the above-mentioned buffer. Six to 10 mg -41 of the material is then applied to a monoQ 10/10 column (Pharmacia) equilibrated in the same buffer. The porin is eluted from a shallow 0.1 to 0.6 M NaCI gradient with a 1.2% increase per min over a 50 min period.

The Flow rate is 1 ml/min. The peak containing porin is collected and dialyzed against TEN buffer and 0.05% 3 ,1 4 -zwittergen. The porin may be purified further by another S-300 chromatography.

Example 6. Purification and chemical modification of the polysaccharides The capsular polysaccharide from both group B Neisseria meningitidis and E. coli K1 consists of a(2-8) polysialic acid (commonly referred to as GBMP or K1 polysaccharide). High molecular weight polysaccharide isolated from growth medium by precipitation (see, Frasch, "Production and Control of Neisseria meningitidis Vaccines" in Bacterial Vaccines, Alan R. Liss, Inc., pages 123-145 (1990)) was purified and chemically modified before being coupled to the porin protein. The high molecular weight polysaccharide was partially depolymerized with 0.1 M acetic acid (7 mg polysaccharide/ml), pH 6.0 to 6.5 (70°C, 3 hrs) to provide polysaccharide having an average molecular weight of 12,000- 16,000. After purification by gel filtration column chromatography (Superdex 200 prep grade, Pharmacia), the polysaccharide was N-deacetylated in the presence of NaBH 4 and then N-propionylated as described by Jennings et al. Immunol. 137:1808 (1986)) to afford N-Pr GBMP. Treatment with NalO 4 followed by gel filtration column purification gave the oxidized N-Pr GBMP having an average molecular weight of 12,000 daltons.

42 Example 7. Coupling of oxidized N-Pr GBMP to the group B meningococcal class 3 porin protein (PP) The oxidized N-Pr GBMP (9.5 mg) was added to purified class 3 porin protein (3.4 mg) dissolved in 0.21 ml of 0.2 M phosphate buffer pH 7.5 which also contained 10% octyl glucoside. After the polysaccharide was dissolved, sodium cyanoborohydride (7 mg) was added and the reaction solution was incubated at 37 0 C for 4 days. The reaction mixture was diluted with 0.15 M sodium chloride solution containing 0.01% thimerosal and separated by gel filtration column chromatography using Superdex 200 PG. The conjugate (N-Pr GBMP-PP) was obtained as single peak eluting near the void volume. Analysis of the conjugate solution for sialic acid and protein showed that the conjugate consists of 43% polysaccharide by weight. The porin protein was recovered in the conjugate in 44% yield and the polysaccharide in 12% yield. The protein recoveries in different experiments generally occur in the 50-80% range and those of the polysaccharide in the 9-13% range.

Example 8. Immunogenicity studies The immunogenicities of the N-Pr GBMP-PP conjugate and those of the N-Pr GBMP-Tetanus toxoid (N-Pr GBMP-TT) conjugate which was 20 prepared by a similar coupling procedure were assayed in 4-6 week old outbread Swiss Webster CFW female mice. The polysaccharide (2 pg)- *conjugate was administered on days 1, 14 and 28, and the sera collected on day 38. The conjugates were administered as saline solutions, adsorbed on aluminum hydroxide, or admixed with stearyl tyrosine. The sera ELISA S 25 titers against the polysaccharide antigen and bactericidal titers against N.

meningitidis group B are summarized in Table 1.

43- Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other perimeters without affecting the scope of the invention or any embodiment thereof.

All patents and publications cited herein are fully incorporated by reference herein in their entirety.

*0.S 0**0 0 -0 44- Table 1.

ELISA and Bactericidal Titers of Group B3 Meningococcal ConjugateVaccines_(N-PrGBMP-Protein) AdjuvantBactericidal Vaccine Adjuvan ELISA Titer Titer N-Pr GBMP-TT Saline 5,400 0 Al(OH) 3 13,000 0 ST' 17,000 0 CFAI 40,000 800 N-Pr GBMP-PP Saline 20,000 500 Saline 22,000 150 Saline 39,000 960

AI(OH)

3 93,000 200

AI(OH)

3 166,000 3,200

AI(OH)

3 130,000 1,200 ST 53,000 1,000 ST 29,000 1,700 ST 72,000 1,500 N-Pr GBMP Saline 100 0 AI(OH)3 100 0 100 0 PP Saline 100 0

AI(OH)

3 100 0 660 0=

S

S

S S. S 5.55

S

1 ST Stearyl tyrosine.

2 CFA Complete Freund's Adjuvant SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The Rockefeller University 1230 York Avenue New York, New York 10021 United States of America North American Vaccine, Inc.

12103 Indian Creek Court Beltsville, Maryland 20705 United States of America INVENTORS: Blake, Milan B.

Tai, Joseph Y.

Qi, Huilin L.

Liang, Shu-Mei Hronowski, Lucjan J.J.

Pullen, Jeffrey K.

(ii) TITLE OF INVENTION: Method for the High Level Expression, Purification and Refolding of the Outer Membrane Group B Porin Proteins from Neisseria Meningitidis (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Sterne, Kessler, Goldstein Fox STREET: 1100 New York Ave., Suite 600 CITY: Washington STATE: D.C.

COUNTRY: USA ZIP: 20005-3934 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: To be Assigned FILING DATE: Herewith

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

Application No.: US 08/096,182 Filing Date: 23 July 1993 )p (viii) ATTORNEY/AGENT INFORMATION: NAME: Esmond, Robert W.

S(B) REGISTRATION NUMBER: 32,893 REFERENCE/DOCKET NUMBER: 1438.006PCOO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (202) 371-2600 TELEFAX: (202) 371-2540 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 930 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..930 i As

G

AC

6

G

Gi'

AC(

Tin

AA(

Asr

TCT

Ser

GAC

Asp 145

TAC

Tyr

CAT

His

COT

Arg

GTA

Val

TCT

Ser 225

ACG

Thr

GCA

Ala (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: C'G TAC GGT ACA ATT AAA OCA GGC GTA GAA ACT TCC ~u Tyr Gly Thr Ile Lys Ala Gly Val Giu Thr Ser 10 ~C CAG AAC GC-C CAA GTT ACT GAA GTT ACA ACC OCT .s Gin Asn Oly Gin Val Thr Gin Val .Thr Thr Ala 25 .TTG GGT TCG AAA ATC GGC TTC AAA GGC CAA GAA p Leu Gly Ser Lys Ile Gly Phe Lys Gly Gin Gin 40 C CTG AMA GCC ATT TGO CAG GTT GAG CAA AAA GCA y Leu Lys Ala Ile Trp Gin Val Gin Gin Lys Ala s0 55 60 T GAC TCC GOT TOO 000 AAC COO CAA TCC TTC ATO r Asp Ser Gly Trp Oly Asn Arg Gin Ser Pile Ile 5 70 75 CTTC GGT AAA TTO COC GTC GOT COT TTG AAC AGC y Phe Gly Lys Leu Arg Val Gly Arg Leu Asn Ser 85' 90 CGOC GAC ATC AAT CCT TGG OAT AGC AAA AGC GAC Gly Asp Ile Asn Pro Trp Asp Ser Lys Ser Asp 100 105 AAA ATT 0CC OAA CCC GAG OCA CGC CTC ATT TCC Lys Ile Ala Gin Pro Oiu Ala Arg Le Ile Ser 115 120 CCC GAA TTT 0CC 0CC CTC AOC GOC AGC OTA CAAJ Pro Oiu Phe Ala Oly Len Ser Oly Ser Vai Gin 130 135 140 AAT OCA GOC AGA CAT AAC AOC OAA TCT TAC CAC G Asn Ala Oly Arg His Asn Ser Gin Ser Tyr His A 150 155.

AA MO GOT GOC TTC TTC OTO CAA TAT 000 GOT G Lys Asn Gly Oly Pile Pile Val Gin Tyr Oiy Gly A 165 170 CAT CAA OTO CAA GAG GOC TTG AAT ATT GAG AAAT His Gin Val Gin Gin Oly Len Asn Ile Gin LysT 180 185 TTO GTC AOC GOT TAC GAC AAT OAT 0CC CTO TAC 0 Len Val Ser Oly Tyr Asp Asn Asp Ala Len Tyr A 195 200 2 CAG CMA CAA GAC OCO AAA CTG ACT OAT OCT TCC A Gin Gin Gin Asp Ala Lys Len Thr Asp Ala Ser A 210 215 220 CAA ACC GAA OTT 0CC OCT ACC TTG GCA TAC COC T Gin Thr Oiu Vai Ala Ala Thr Len Ala Tyr Arg P3 230 235 CCC CGA OTT TCT TAO 0CC CAC GOC TTC AMA GOT T Pro Arg Val Ser Tyr Ala His Gly Pile Lys Oly L 245 250 GAO ATA 000 AAC GMA TAC GAC CMA OTO OTT OTO GC Asp Ile Oly Asn Gin Tyr Asp Gin Val Val Vai 0] 260 265 COC TCT OTA TTT Arg Ser Val Phe ACC GOC ATC OTT Thr Gly Ile Vai GAO OTO GOT AAC Asp Len Gly Asn TOT ATC 0CC GOT Ser Ile Ala Giy 000 TTG AAA GOC Olyi Len Lys Oly OTO OTO AMA GAC Val Len Lys Asp TAT TTG GOT OTA Tyr Len Gly Vai 110 3TA COO TAO OAT 7l Arg Tyr Asp ['AC 000 OTT AAC .yr Ala Len Asn CC 000 TTC AAC la Oly Phe Asn 160 COTAT AAA AGA la Tyr Lys Arg 175 AC CAG ATT CAC yr Gin Ile His 190 CT TOO OTA 000 la Ser Val Ala PT TOG CAC AAO sn Ser His Asn TO 000 AAC OTA aie Oly Asn Val 240 OTT OAT OAT mn Val Asp Asp 255 T 000 GMA TAO y~ Ala Gin Tyr 270 48 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 GAC TTC TCC AAA CGC ACT TCT GCC TTG GTT TCT GCC GGT TGG TTG CAA 864 Asp Phe Ser Lys Arg Thr Ser Ala Leu Val Ser Ala Gly Trp Leu Gin 275 280 285 GAA GGC AAA GGC GAA AAC AAA TTC GTA GCG ACT GCC GGC GGT GTT GGT 912 Glu Gly Lys Gly Glu Asn Lys Phe Val Ala Thr Ala Gly Gly Val Gly 290 295 300 CTG CGT CAC AAA TTC TAA Leu Arg His Lys Phe 930 305 310 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 309 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Leu Tyr Gly Thr Ile Lys Ala Gly Val Glu Thr Ser Arg Ser Val Phe 1 5 10 His Gln Asn Gly Gin Val Thr Glu Val Thr Thr Ala Thr Gly Ile Val 25 Asp Leu Gly Ser Lys Ile Gly Phe Lys Gly Gin Glu Asp Leu Gly Asn 40 Gly Leu Lys Ala Ile Trp Gin Val Glu Gin Lys Ala Ser Ile Ala Gly 55 Thr Asp Ser Gly Trp Gly Asn Arg Gin Ser Phe Ile Gly Leu Lys Gly 70 75 Gly Phe Gly Lys Leu Arg Val Gly Arg Leu Asn Ser Val Leu Lys Asp 85 90 Thr G l y Asp Ile Asn Pro Trp Asp Ser Lys Ser Asp Tyr Leu Gly Val 100 105 110 Asn Lys Ile Ala Glu Pro Glu Ala Arg Leu Ile Ser Val Arg Tyr Asp 115 120 125 Ser Pro Glu Phe Ala Gly Leu Ser Gly Ser Val Gln Tyr Ala Leu Asn .".130 135 140 Asp Asn Ala Gly Arg His Asn Ser Glu Ser Tyr His Ala Gly Phe Asn 145 150 155 160 Tyr Lys Asn Gly Gly Phe Phe Val Gin Tyr Gly Gly Ala Tyr Lys Arg 165 170 175 His His Gin Val Gin Glu Gly Leu Asn Ile Glu Lys Tyr Gin Ile His 180 185 190 Arg Leu Val Ser Gly Tyr Asp Asn Asp Ala Leu Tyr Ala Ser Val Ala 195 200 205 Val Gin Gin Gin Asp Ala Lys Leu Thr Asp Ala Ser Asn Ser His Asn 210 215 220 Ser Gin 225 Thr Pro Ala Asp Asp Phe Glu Gly 290 Leu A-rq 305 Thr Glu Arg Val Ile Gly 260 Ser Lys 275 Lys Gly His Lys Val Ala Ala Thr Leu Ala Tyr Arg Phe Gly Asn Val 230 235 240 Ser Tyr Ala His Gly Phe Lys Gly Leu Val Asp Asp 245 250 255 Asn Glu Tyr Asp Gin Val Val Val Gly Ala Glu Tyr 265 270 Arg Thr Ser Ala Leu Val Ser Ala Gly Trp Leu Gin 280 285 Glu Asn Lys Phe Val Ala Thr Ala Gly Gly Val Gly 295 300 Phe INFORMATION FOPR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1029 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 1.-1029 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG GAC GTT ACC Met Asp Val Thr TTG TAC GGT ACA ATT AAA Leu Tyr Gly Thr Ile Lys GCA GGC GTA GAA GTT TCT Ala Gly Vai Giu Val Ser

*SSS

S

S

1

CGC

Arg

ACT

Thr

CAA

Gin

AAA

Lys

TTC

Phe

AAC

Asn

GTA

Val

GCA

Ala.

OAA

Olu 50

GCC

Ala

ATC

Ile

ACC

Thr

AAA

Lys

ACC'

Thr 35

GAC

Asp

TCC

Ser

GGC

Gly

GTA

Val

GAT

Asp :.20 Gin'

CTC

Leu

ATC

Ile

TTG

Leu

TTG

Leu 100

GCT

Ala

ATT

"Ile

GC

Gly

GCC

Ala

AAA

Lys 85

AAA

Lys

GGT

Giy

CC

Ala

AAC

Asn

GGC

Gly 70

GGC

Gly

GAC

Asp

ACA

Thr

GAC

Asp

GGC

Gly 55

ACT

Thr

GGC

Gly

AGC

Ser

TAT

Tyr

TTC

Phe 40

ATG

Met

AAC

Asn

TTC

Phe

GGC

Gly

CTG

Leu 12 0

AAA

Lys 25

GGT

Gly

AAA

Lys

AGC

Ser

GGT

Giy

GAC

Asp 105

GGA

Gly 10

OCT

Aia

TCT

Ser

GCC

Ala

GGC

Gly

ACC

Thr 90

AAC

Asn

CTG

Leu CAA GGC GGA Gin'Giy Gly AAA ATC GGT Lys Ile Gly 45 ATT TGG CAG Ile Trp Gin 60 TGG GGT AAC Trp Oly Asn 75 GTC CGC GCC Val Arg Ala OTC AAT GCA Val Asn Ala GGT ACT ATC Gly Thr Ile 125

AAA

Lys 30

TTC

Phe

TTG

Leu

CC

Arg

GGT

Gly

TGG

Trp

GGT

Gly

TCT

Ser

AAA

Lys

GAA

diu

CAG

Gin

AAT

Asn

GAA

Giu

CGT

Arg

AAA

Lys

GGT

Gly

CAA

Gin

TCC

Ser

CTG

Leu

TCT

Ser

GTA

Val 144 i192 240 288 336 384 GOT TCT AAC ACC Gly Ser Asn Thr GAA OAT GTA Glu Asp Val GAA. AGC CGT GAA ATC TCC GTA CGC TAC GAC TCT Glu Ser 130 Arg Glu Ile Ser Val 135 Arg Tyr Asp Ser CCC GTA TTT GCA GGC Pro Val Phe Ala Gly TTC AGC GGC AGC GTA Phe Ser Gly Ser Val 145 GAT AAA TAC AAA CAT Asp Lys Tyr Lys His 165

CAA

Gin 150 TAC GTT CCG CGC Tyr Val Pro Arg AAT GCG AAT GAT Asn Ala Asn Asp ACG AAG TCC AGC Thr Lys Ser Ser GAG TCT TAC CAC Glu Ser Tyr His GCC GGT Ala Gly 175 CTG AAA TAC Leu Lys Tyr GCC AAA TAT Ala Lys Tyr 195 AAT GCC GGT TTC Asn Ala Gly Phe GGT CAA TAC GCA Gly Gin Tyr Ala GGT TCT TTT Gly Ser Phe 190 GCA GTA AAT Ala Val Asn GCT GAT TTG AAC ACT GAT GCA GAA CGT Ala Asp Leu Asn Thr Asp Ala Glu Arg 200 ACT GCA Thr Ala 210 AAT GCC CAT CCT GTT AAG GAT TAC CAA Asn Ala His Pro Val Lys Asp Tyr Gin 215

GTA

Val 220 CAC CGC GTA GTT His Arg Val Val GGT TAC GAT GCC Gly Tyr Asp Ala GAC CTG TAC GTT Asp Leu Tyr Val GTT GCC GGT CAG Val Ala Gly Gin

TAT

Tyr 240 GAA GCT GCT AAA Glu Ala Ala Lys

AAC

Asn 245 AAC GAG GTT GGT Asn Glu Val Gly

TCT

Ser 250 ACC AAG GGT AAA Thr Lys Gly Lys AAA CAC Lys His 255 GAG CAA ACT Glu Gin Thr ACG CCT CGC Thr Pro Arg 275

CAA

Gin 260 GTT GCC GCT ACT Val Ala Ala Thr GCT TAC CGT TTT Ala Tyr Arg Phe GGC AAC GTA Gly Asn Val 270 GTG AAT GGC Val Asn Gly GTT TCT TAC GCC Val Ser Tyr Ala GGC TTC AAA GCT Gly Phe Lys Ala

AAA

Lys 285 GTG AAA Val Lys 290 GAC GCA AAT TAC Asp Ala Asn Tyr TAC GAC CAA GTT Tyr Asp Gin Val

ATC

Ile 300 GTT GGT GCC GAC Val Gly Ala Asp 912

TAC

Tyr 305 GAC TTC TCC Asp Phe Ser AAA CGC Lys Arg 310 ACT TCC GCT CTG GTT TCT GCC GGT TGG Thr Ser Ala Leu Val Ser Ala Gly Trp 315 *c AAA CAA GGT AAA Lys Gin Gly Lys GGT CTG CGT CAC Gly Leu Arg His 340 GCG GGA AAA GTC Ala Gly Lys Val CAA ACT GCC AGC Gin Thr Ala Ser ATG GTT Met Val 335 1008 1029 AAA TTC TAA Lys Phe INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 342 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Asp Val Thr Leu Val Lys Asp Ala so DESCRIPTION: SEQ ID NO:4: Tyr Gly Thr Ile Lys Ala Gly 10 Gly Thr Tyr Lys Ala Gin Gly 25 Vai Gly Val Ser Ser Lys Thr Ala Thr Gin Ile Aia Asp Phe Gly Ser LVs Ile Gin Giu Asp Leu Gly Asn so 40 Gly Met Lys 55 Thr Asn Ser Ala Ile Trp Giy Trp Giy Gly Phe Lys Gin Leu Giu Asn Arg Gin Gly Gin Lys Phe Asn Giy Giu Phe 145 Asp Leu Ala Thr Ala 225 Glu Glu Ala Ser Ile Alz Ile Thr Ser Ser 130 Ser Lys Lys Lys Ala 210 Gly Ala Gln Gly *Vai *Asn 115 Arg Gly Tyr Tyr Tyr 195 Asn Tyr.

Ala Thr Leu Leu 100 Thr Giu Ser Lys Glu 180 Al a Ala Asp L~ys Gln 260 Lys Lys Giu Ile Vai His 165 Asn Asp His Ala 245 Val Gly 70 Gly Asp Asp Ser Gin 150 Thr Ala Leu Pro Asn 230 Asn Ala Gly Ser Val Val 135 Tyr Lys Gly Asn Val 215 Asp Glu ka 75 Phe Gly Thr Val Arg Ala Gly Asn Leu 90 Gly Asp Asn Val Asn Ala Trp Glu Ser 105 110 Leu Gly Leu Giy Thr Ile Gly Arg Val 120 125 Arg Tyr Asp Ser Pro Val Phe Ala Gly 140 Val Pro Arg Asp Asn Ala Asn Asp Val 155 160 Ser Ser Arg Giu Ser Tyr His Ala Gly 170 175 Phe Phe Gly Gin Tyr Ala Gly Ser Phe 185 190 Thr Asp Ala diu Arg Val Ala Val Asn 200 205 Lys Asp Tyr Gin'Val His Arg Val Val .220 Leu Tyr Val Ser Val Ala Gly Gin Tyr 235 240 Val Giy Ser Thr Lys Gly Lys Lys His 250 255 Thr Ala Ala Tyr Arg Phe Gly Asn Val 265 270 His Gly Phe Lys Ala Lys Val Asn Gly 280 285 a.

a.

Thr Pro Arg Val Ser Tyr 275 Ala a.

a .aa.

Val Tyr 305 Lys Gly Lys Asp 290 Asp Phe Gin Gly Leu Arg Ala Ser Lys His 340 Tyr Arg 310 Ala Phe Gin Tyr Asp 295 Thr Ser Ala Gly Lys Val Gin Val Leu Val 315 dlii Gin 330 Val Ala Ala Gly Ala Gly Trp Ser Met 335 INFORMATION FOR SEQ ID SEQUJENCE

CHARACTERISTICS:

LENGTH: 1.092 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1.-1092 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GCT AGC ATG ACT GGT OGA CAG CAA ATG GGT CGG Met Ala Ser Met Thr Gly Gly Gin Gin Met Gly Arg 10 GTA CCC AGC TCG GAT CCA GAC GTT ACC TTG TAC GGT Val Pro Ser Ser Asp Pro Asp Val Thr Leu Tyr Gly 25 GGC GTA GAA GTT TCT CGC GTA AAA GAT OCT GOT ACA Oly Val Oiu Val Ser Arg Val Lys Asp Ala Oly Thr 40 GGC OCA AAA TCT AAA ACT GCA ACC CAA ATT GCC GAC Gly Oly Lys Ser Lys Thr Ala Thr Gin Ile Ala Asp 55 60 ATC OCT TTC AAA GOT CAA GAA GAC CTC GGC AAC GGC Ile Gly Phe Lys Gly Gin Ciu Asp Leu Gly Asn Oly 70 75 TGG CAG TTG CAA CAA AAA CCC TCC ATC GCC GGC ACT Trp Gin Leu Giu Gin Lys Ala Ser Ile Ala Oly Thr 90 CGT AAC CCC CAC TCC TTC ATC GGC TTC AAA GGC GGC Cly Asn Arg Gin Ser Phe Ile Cly Leu Lys Oly Giy 100 105 CGC CCC GGT AAT CTG AAC ACC OTA TTG AAA GAC AGC C Arg Ala Gly Asn Leu Asn Thr Val Leu Lys Asp Ser C 115 120 1 AAT OCA TOG CAA TCT GGT TCT AAC ACC GAA GAT GTA C Asn Ala Trp Giu Ser Oly Ser Asn Thr Giu Asp Val L 130 135 140 ACT ATC GGT COT GTA GAA AGC COT GAA ATC TCC GTA C Thr Ile Gly Arg Val Giu Ser Arg GiuIle Ser Val 145 150 i55 CCC OTA TTT OCA GGC TTC AGC GGC AGC GTA CAA TAC G Pro Vai Phe Ala Cly Phe Ser Oly Ser Val Gin Tyr V 165 170 AAT CC AAT CAT CTC CAT AAA TAC AAA CAT ACO AAG T( Asn Ala Asn Asp Vai Asp Lys Tyr Lys His Thr Lys SE 180 185 TCT TAC CAC GCC GGT CTG AAA TAC GAA AAT GCC GGT TJ Ser Tyr His Ala Oly Leu Lys Tyr Giu Asn Ala Cly Pf 195 200 20 a.

a. a a a a

G)

AC

TA

Ty 4

TT

Ph

AT

Me ks2

'T(

?hE ;ly

~TG

eu

C

rg

TT

al er le .T TCA AGC TTG sp Ser Ser Leu M ATT AAA OCA "r Ile Lys Ala rAAA GCT CAA *r Lys Ala Gin C GGT TCT AAA e Oly Ser Lys G AAA CCC ATT t Lys Ala Ile C AGC GCC TG I Ser Oly Trp OCT ACC GTC Gly Thr Val 110 GAC AAC OTC -Asp Asn Val GGA CTG GOT Gly Leu Oly TAC CAC TCT Tyr Asp Ser 160 CCG CCC CAT Pro Arg Asp 175 AGC CGT GAG Ser Arg Giu 190 TTC GOT CAA Phe Gly Gin 144 192 240 288 336 384 432 480 528 576 624 52

TTTG

p Leu TAC GCA GOT TCT TTT 0CC Tyr Ala Gly Ser Phe Ala 210 ,AAZA TAT OCT GA

CGT

Arg 225

OTA

Val

GTT

Val

AAG

Lys

CGT

Arg

GCT

Ala 305

ATC

Ile

OTT

Val

CAC

His

GCC

Ala

GGT

Gly

TTT

Phe 290

AAA

Lys

OTT

Val1

GCA

Ala

CC

Arg

GT

Gly

AAA

Lys 275

OGC

Gly

GTG

Val

GOT

Gly

OTA

Val

OTA

Val1

CAO

Oin 260

AAA

Lys

AAC

Asn

AAT

Asn 0CC Ala

AAT

Asn

OTT

Val 245

TAT

Tyr

CAC

His

OTA

Val

OOC

Oly

GAC

Asp 325

ACT

Thr 230 0CC Ala

GAA

Oiu

GAO

Olu

ACO

Thr

OTO

Vai 310

TAC

Tyr Lys 215

OCA

Ala

GOT

Oly

OCT

Ala

CAA

Gin

CCT

Pro 295

AAA

Lys

GAC

Asp Tyr Ala As AAC ACT OAT GCA OAA Asn Thr Asp Ala Glu AAT 0CC Asn Ala TAC OAT Tyr Asp OCT AAA Ala Lys 265 ACT CAA Thr Gin 280 CG OTT Arg Val GAC OCA Asp Ala TTC TCC Phe Ser

CAT

His 0CC Ala 250

AAC

Asn

OTT

Val

TCT

Ser

AAT

Asn

AAA

CCT OTT Pro Val 235 AAT GAC Asn Asp AAC GAO Asn Giu 0CC OCT Ala Ala TACG0CC Tyr Ala 300 TAG CAA Tyr Gin 315 CG ACT Arg Thr

AAG

Lys

CG

Leu

OTT

Val

ACT

Thr 265

CAC

His

TAC

Tyr

TCG

Ser OAT TAG Asp Tyr TAC OTT Tyr Val 255 GOT TCT Gly Ser 270 0CC OCT Ala Ala GOC TTC Gly Phe GAG CAA Asp Gin OCT CTO Ala Leu

CAA.

Gin 240

TCT

Ser

ACC

Thr

TAC

Tyr

AAA

Lys

OTT

Val1 320

OTT

720 768 816 864 912 960 1008 1056 1092 330 335 TCT 0CC Ser Ala GOT TOO Gly Trp 340 TTO AAA CAA GOT AAA GOC OCO OOA AAA OTC OAA CAA Leu Lys Gin Oly Lys Oly Ala Oly Lys Val Glu Gin 345 350 9*

S

S. a ACT 0CC AOC ATO OTT GOT CTO COT CAC AAA TTC TAA Thr Ala Ser Met Val Gly Leu Arg His Lys Phe 355 360 INFORMATION FOR SEQ ID NO:6: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 363 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ala Ser Met Thr Oly Oly Gin Gin Met Gly Arg Asp Ser Ser Leu 1 5 10 Val Pro Ser Ser Asp Pro Asp Val Thr Leu Tyr Oly Thr Ile Lys Ala 20 25 Oly Val Glu Val Ser Arg Val Lys Asp Ala Gly Thr Tyr Lys Ala Gin 40 Gly Gly Lys Ser Lys Thr Ala Thr Gin Ile Ala Asp Phe Gly Ser Lys 55 Ile Gly Phe Lys Oly Gin Oiu Asp Leu Gly Asn Gly Met Lys Ala Ile 70 75 B0 Trp Gin Leu Giu Gin Lys Ala Ser Ile Ala Gly Thr Asn Ser 90 Gly Trp Gly Asn Arg Ala Asn Ala 130 Thr Ile 145 Pro Val Asn Ala Ser Tyr Tyr Ala 210 Arg Val 225 Val His Val Ala Lys Gly Arg Phe 290 Ala Lys 305 Ile Val Ser Ala Thr Ala Arg Gly 115 Trp Gly Phe Asn His 195 Gly Ala Arg Giy Lys 275 Gly Val Gly Gly Ser 355 Gin 100 Asn Glu Arg Ala Asp 180 Ala Ser Val1 Val Gin 260 Lys Asn Asn Al a Trp, 340 Met Ser Leu Ser Val1 Gly 165 Val Gly Phe Asn Val1 245 Tyr His Val1 Giy Asp 325 Leu Val1 Phe Asn Gly Glu 150 Phe Asp Leu Ala Thr 230 Ala Glu Giu Thr Val1 310 Tyr Lys Gly Ile Thr Ser 135 Ser Ser Lys Lys Lys 215 Ala Gly Ala Gin Pro 295 Lys Asp Gin Leu Gly Val 120 Asn Arg Gly Tyr Tyr 200 Tyr Asn Tyr Ala Thr 280 Arg Asp Phe Gly Arg 360 Leu 105 Leu Thr Giu Ser Lys 185 Giu Ala Ala Asp Lys 265 Gin Val Ala Ser Lys 345 His Lys Lys Glu Ile Val 170 His Asn Asp His Ala 250 Asn Val1 S er Asn Lys 330 Gly Lys Gly Asp Asp Ser 155 Gin Thr Ala' Leu Pro 235 Asn Asn Ala Tyr Tyr 315 Arg Ala Phe Gly Phe Gly 110 Ser Gly Asp .125 Val Leu Gly 140 Val Arg Tyr Tyr Val Pro Lys Ser Ser 190 Gly Phd Phd 205 Asn Thr Asp 220 Val Lys Asp Asp Leu Tyr Giu Val Gly 270 Ala Thr Ala 285 Ala His Gly 300 Gin Tyr Asp Thr Ser Ala Gly Lys Val 350 Thr Asn Leu Asp Arg 175 Arg Gly Ala Tyr Val 255 Ser Ala Phe Gin Leu 335 Glu Val Val Gly Ser 160 Asp Giu Gin Glu Gin 240 Ser Thr Tyr Lys Val 320 Val Gin .9

S

*9 9. *99* INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 187 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 101. .187 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG AGACCACAAC GGTTTCCCTC TAGAAATAAT TTTGTTTAAC TTAAAGAAGG AGATATACAT ATG GCT AGC ATG ACT Met Ala Ser Met Thr 1 GGT GGA CAG CAA ATG GGT CGG GAT TCA AGC TTG GTA CCG AGC TCG GAT Gly Gly Gin Gin Met Gly Arg Asp Ser Ser Leu Val Pro Ser Ser Asp 15 CTG CAG GTT ACC TTG TAC GGT ACA Leu Gin Val Thr Leu Tyr Gly Thr 115 163 187 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Ser Met Thr Gly Gly Gin Gin Met 1 5 10 Val Pro Ser Ser Asp Leu Gin Val Thr Leu INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 54 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both NO:8: Gly Arg Asp Ser Ser Leu Tyr Gly Thr (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTT GGT CTG CGT CAC AAA TTC TAACTCGAGC AGATCCGGCT GCTAACAAAG Val Gly Leu Arg His Lys Phe 1

CCC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Val Gly Leu Arg His Lys Phe 1 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGGTAGATC TGCAGGTTAC CTTGTACGGT ACAATTAAAG CAGGCGT 47 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION:.SEQ ID NO:12: GGGGGGGTGA CCCTCGAGTT AGAATTTGTG ACGCAGACCA AC 42 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCAAGCTTGG TACCGAGCTC INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TTTGTTAGCA GCCGGATCTG INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID CTCAAGACCC GTTTAGAGGC C 21 S(2) INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AGCGGCTTGG AATTCCCGGC TGGCTTAAAT

TTC

33 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CAAACGAATG AATTCAAATA

AAAAAGCCTG

INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCTGTTGCAG CACATATGGA CGTTACCTTG TACGGTACAA TTAAAGC 47 INFORMATION FOR SEQ ID NO:19: SEQUENCE

CHARACTERISTICS:

LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CGACAGGCTT TTTCTCGAGA CCAATCTTTT CAG INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear o* (xi) SEQUENCE DESCRIPTION: SEQ ID CCTGTTGCAG CGGATCCAGA CGTTACCTTG TACGGTACAA TTAAAGC 47 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGACAGGCTT TTTCTCGAGA CCAATCTTTT CAG 33 INFORMATION FOR SEQ ID NO:22: 57 SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTTGCAGCAC ATATGGACGT TACCCTGTAC GGCACC 36 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGGGGGATGG ATCCAGATTA GAATTTGTGG CGCAGACCGA CACC 44

S.

Claims (9)

1. A method of refolding a recombinantly produced outer membrane meningococcal group B porin protein or fusion protein thereof, comprising: lysing transformed E. coli host cells capable of expressing the mature meningococcal group B porin protein or fusion protein thereof to release the protein as insoluble inclusion bodies; washing said insoluble inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; suspending and dissolving said inclusion bodies in an aqueous °solution of a denaturant; diluting said solution with a detergent, with the proviso that said detergent is not SDS; and S. passing said diluted solution through a gel filtration column; whereby folded, trimeric protein is obtained.

2. The method of claim 1, wherein the diluted solution obtained in step has a concentration of less than 10 mg protein/ml.

3. A substantially pure outer membrane meningococcal group B porin protein or fusion protein thereof produced according to the method of claim 1.

4. The substantially pure protein of claim 3, which is the mature group B class 2 porin protein. The substantially pure protein of claim 3, which is the mature group B class 3 porin protein. 29/7/99 -1 1111111111111110 59

6. A substantially pure refolded outer membrane meningococcal group B porin protein or fusion protein thereof produced according to the method of claim 1.

7. A vaccine comprising the outer membrane meningococcal group B porin protein or fusion protein thereof of claim 3 or claim 6 together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein said protein is present in an amount effective to elicit protective antibodies in an :0 animal to Neisseria meningitidis.

8. The vaccine according to claim 7, wherein said protein is conjugated S" to a Neisseria meningitidis capsular polysaccharide.

9. A method of obtaining a meningococcal group B porin protein or fusion protein-polysaccharide conjugate comprising: obtaining the refolded protein according to the method of claim 1; obtaining a Neisseria meningitidis capsular polysaccharide; and conjugating the protein to the polysaccharide of The method of claim 9, wherein said protein has the amino acid sequence depicted in Figure 4.

11. A method of preventing bacterial meningitis in an animal comprising administering to said animal the meningococcal group B porin protein or 29/7/99 fusion protein thereof produced according to claim 1 in an amount effective to prevent bacterial meningitis. Dated this 14 day of July, 1998. THE ROCKEFELLER UNIVERSITY AND NORTH AMERICAN VACCINE, INC Patent Attorneys for the Applicants PETER MAXWELL ASSOCIATES So oo*oo a o*