CA2111110A1 - Production and recovery of recombinant neurotrophins - Google Patents

Abstract

We disclose a process for producing and recovering a recombinant neurotrophin comprising the steps of: (1) culturing a non-animal host cell transformed with a recombinant DNA molecule comprising an expression control sequence operatively linked to a DNA
sequence encoding a neurotrophin; (2) solubilizing the neurotrophin in a solution comprising a protease inhibitor in an amount effective to inhibit degradation of the neurotrophin by proteases and a strong denaturing agent, said solution being essentially free of reducing agents; (3) exchanging the strong denaturing agent for a weak denaturing agent; (4) adjusting the solution to comprise a basic amino acid at a concentration effective to maintain the solubility of the neurotrophin in a nondenaturing environment; (5) removing the weak denaturing agent; and (6) purifying the neurotrophin. In a specific embodiment, also disclosed is the bacterial production of recombinant neurotrophin-4.

Description

1~ 20AUG1993 tlllo PcTlus 92 /05 o o PRODUCTION AND RECOVERY OF
RECOMBINANT NEUROTROPHINS
This application is a continuation-in-part of copending applic~tion Serial No. 07/715,1~5, filed June 12, 1991, which is incorporated by referance in its entirety.

1. INTRQDUCTION
This invention relates to the fields of neurobiology, molecular biology and protein biochemistry and, in particular, to neurotrophins and methods o~ produc:ing ~nd recovering them.

2. BACKGROUND ~ THE: INYENTION
The neurotrophins ars a group of proteins in~olved in the functioning of the nervous system. They stimulate the growth of nerve cells and, during embryonic dev lopment, cupport their survival. To date, researchers have id~ntified four neurotrophins: nerve growth factor (NGF) (Ullrich et al., 1983, Nature ~03:821-825), brain derived neurotrophic factor (BDNF) (Leibrock et al.; 1989, Nature 341:149-152), naurotrophic factor 3 (NT-3) (Maisonpierre et al~, 1990, Sr-i~nce .~47:1446-1451) and neurotrophin-4 ~Hallbook, et al.; 1991, Neuron 6:845-8s8~. Nerve growth factor is e sential for the development and survival of the peripheral and sympathetic nervous system and the cholinergi~ neurons of the brain. It is currently being tested for u~e in Al2heimer's disease. ~DNF promotes the survival of sensory neurons in the central nervous ~ystem and shows promise as a therapy for Parkinson's disease.
NT-3 and NT-4 are newly discovered and their biological role is now baing investigated. Like NGF, NT-3 seems to act on ~ensory and sympathetic neuron~. -Native human N~F has three subunits~ and ~. Only the B subunit has neurotrophic activity. ~-NGF is produced lP~yUS 2 ~A~(lG 1~93~
-2lllllo ~CT~s92/o5oo6!

as a pre-pro-protein which is secreted from the cell and processed into the mature form. The pre-pro-peptide contains 187 amino acid residues. During processing, the pre- and the pro-sequences are removed, as are two amino acid residues at the C-terminus. This yiel~s a mature protein of 118 amino acid residues. The mature protein has six cysteine residues that form three disulfide bonds. The protein also contains many basic amino acids resulting in a positive charge at neutral pH.
Structurally, the n~urotrophins form a family, apparently having evolved from a common ancsstral gene.
(Hyman et al., 1991, WO 91/03568.) For example, the amino acid sequence of the ~ polypeptide of human NGF thN&F) is 90% homologous to mouse NGF and bovine NGF; and the threQ
human neurotrophins, hNGF, hBDNF and hNT-3, share 60%
homology at the amino acid level. The six cysteine residues are conserved in all known neurotrophins, implying that the disulfide bonds are important for function.
Natural sources of neurotrophins are limited. The mouse submaxillary gland is rich in NGF but isolation is difficult and the quantities recovered, small (Hyman et al., 1991, WO 91/03568). del la Valle et al. (EP 0 333 574, 1989j have reported the isolatisn of small amounts of hNGF from human placenta by chromatographic fractionation.
ThereforQ,-re~earchers have turned to molecular genetics to provide a rea~y source of biologically active neu~otrophins. ~NA sequences encoding NGF, BDNF and NT-3 have all been isolated (Ullrich et al., Nature 3Q3:821-825;
Hyman et al., W0 91/03568; Hohn et al., WO 91/03569; and Kaisho et al., FEBS Letters 266:187-191). Researchers havQ
transformed animal and non-animal hosts with these sequences in order to express the neurotrophins.
Kanaya et al. (1989, Gene 83:65-74) fused a DNA
~equence encoding mature hNGF to one encoding the yeast a-mating factor pre-pro sequence. Upon expression, the WO 92/2266~ PCl`tUS92/0~006 ~lllllO

culture supernatant contained a protein recognized by ~-hNGF antibodies. However, the partially purified protein exhibited low neurotrophic activity. Furthermore, the level of expression was low. Kanaya et al. stated that these results may have been due either to the failure of the yeast system to remove the two extra C-terminal amino acid residues during maturation or to a problem with folding the hNGF protein.
Chan et al. (EP 0 370 171, 1990) produced mature hNGF
in insect cells. They fused a gene for pre-pro-hNGF to the polyhedrin promoter and leader cequence o~ baculoviral DNA.
They reported the production of 6 ~g/ml hNGF, but did not~
physically charasterize the product.
Researchers have also expressed human NGF, BDNF and NT-~ in mammalian e~pression systems. Bruce and Heinrich (1989, Neurobiology of Aging 10:89-94) expressed a DNA
sequence encoding the complete precursor for hNGF in COS
cells and detected hNGF dimer in the conditioned medium.
However, they could not determine the efficiency at which pre-pro-hNGF was converted to mature hNGF. Kakinuma et al.
(EP 0 414 lS1, l991) expressed active hNGF in CHO cells, Hyman et al. (Wo 91/03568, 1991~ expressed hBDNF in CHO
cells. Nakahama et al. (EP 0 386 752, 1990) and ~ohn et al. (WO 91/03569, 1~91) expressed hNT-3 in COS cells.
Mammalian systems provide the most natural environment for the production of mammalian proteins. However, the production of large quan~ities of proteins in these systems is very expensive. Therefore, there is a need to develop systems that are both less expensive and more productive.
One such system is ~. ~Qli-R~searchers have attempted to produce neurotrophins inE. ÇQli, but without signif icant success. Gray and Ullrich tEP O 121 338, 1984) constructed an expression vector `
encoding N-methionyl-hNGF and expressed the gene in E. coli. They reported identification of hNGF by SUBSTITUTE SHEET

W092~22665 PCT/VS92tO~006 ~ 1 10 _ 4 _ immunodetection on Western blot, but they did not isolate the protein or demonstrate biological activity.
Hu and Neet (1988, Gene:57-65) attempted to express mouse NGF in ~. ÇQ1i- They cloned a DNA sequence encod.ng a mature mouse NGF in which they replaced serine, the N--terminal amino acid in the native protein, with methionine.
They inserted the DNA sequence into a plasmid having a temperature inducible lambda PL promoter. This system expresses other heterologous proteins at rates of 10% - 25%
total cellular protein. They expressed the gene and isolated NGF by ammonium sulfate precipitation followed by dialysis against acetate buffer. However, as tested by bioassay, this system yielded only 0.0005% to 0.1% NGF.
The authors speculated that the highly inconsistent and low yields were due to toxicity of NGF to the cells, instability or translational inefficiency of the mRNA, or mismatched disulfide bonds in the refolded, oxidized protein.
Iwai et al~ (1986, Chem. Pharm. Bull. ~4:4724-4730) reported synthesis of a gene encoding hNGF with codons preferred in ~. ÇQ~ hey expressed the gene directly as N-methionyl-hN&F or as a fusion protein with human growth hormone. Direct expression was only one-fourth as efficient as expression of the fusion protein. They examined the proteins by SDS-PAGE, but did not otherwise isolate them.
Dicou et al. (1989, J. Neurosci. Res. 22:13-19) expresssd ~ouse pre-pro-NGF and a fragment of hNGF as fusion proteins with ~-galactosidase and found that the addition of protease inhibitors improved yield.
In summary, previous attempts to express neurotrophins in ~. Qli have resulted in cell death, low levels of protein accumulation, and expression of protein with -virtually no biological activity.

SUBSTITUTE SHEET

WO92~2266~ PCT/US92~050~6 S~; l t ~

3. SUMMARY OF_THE INVENTIo~
This invention provides processes for producing and recovering biologically active recombinant neurotrophins from non-animal host c~lls. Processes for producing recombinant neurotrophins comprise the step of culturing a host cell transformed with a recombinant DNA molecule comprising an expression control sequence operatively linked to a DNA sequence encoding a neurotrophin. When the neurotrophins are expressed directly into the cytop:Lasm, the host cells preferably are protease-deficient mu1;ants and, according to the best mode of which we are aware, heat shock regulatory gene mutants. In bacterial hosts, the neurotrophin gene is preferably under the control of a controllable promoter and expression is preferably un-induced or repressed un~il the cells reach late log phase. The neurotrophins may be also secreted from the cell during production by pro~iding a DNA sequence encoding a signal peptide upstream of the DNA sequence encoding the neurotrophin.
Processes for recovering biologically active . reccmbinant neurotrophins produced by host cell culture~
comprise the step of solubilizing the neurotrophin in a solution comprising a strong dena~uring agent, the solution b~ing essentially free of reducing agen~s. Ac~ording to one embodiment of the invention the solution further comprises a protease inhibitor in an amount effe~tive to inhibit degradation of the neurotrophin by proteases.
Other ~mbodiments o~ the inven~ion ~urther comprise .~ome or all of the following steps: exchanging the strong denaturing agent for a weak denaturing agent; adjusting the solution to co~prise a basic amino acid or equivalent at a concentration effective to maintain solubility of the neurotrophin in a non-denaturing environment; purifying the neurotrophin from other molecules in ~he solution; removing SUBSTITUTE SHEET

W092/2266~ PCT/US92/0~006 ~lllllU - 6 -the weak denaturing agent; and completing the purification in the absence of the denaturing agent.
In a further embodiment o~ the invention, the neurotrophin molecule expressed in E. ~Qli and recovered in a biologically active form is neurotrophin-4. In a preferred embodiment of the invention directed to expression of biologically active NT-4 in E. coli, the NT-4 is encoded by a nucleic acid molecule comprising a sequence substantially as set forth for hNT-4 in Figure 11 (SEQ ID
NO: 22) or may comprise a sequence that is at least about seventy percent homologous to such a sequence.

4. DESCR~PTION OF TH~ DRAWINGS
Figure 1 is a schematic representation of plasmid pRPN133. The solid line represents pBR322-derived DNA
sequences with th~ origin of replication (ORI~ and the ~-lactamase gene (ampicillin resistance~ (Ap) indicated.
Distinctive features of the plasmid are indicated by boxed regions with arrowheads indicating the direction of transcription or replication. PL indicates the lambda PL .
promoter. rbsl is the wild type promoter and ribosome binding site of phage T7 ~1.1. The h~GF gene encodes a mature polypeptide in which the se~ond amino acid residue, serine is replaced by threonine. cI857 indicates the heat inactivatable A repressor gene.
Figure 2 shows dose-respon~e curves to recombinant human NGF produced in E. co~i by (A~ E8 chick embryo dorsal root ganglia (DRG) explants and (B) diæsociated E8 DRG.
Figure 3 depicts the nucleotide and amino acid sequences of wild type LamB (Fig. 3A, SEQ ID NO:l) and the synthetic LamB signal sequences, LamBl (Fig. 3B, SEQ ID
NO:2), LamB2 ~Fig. 3C, SEQ ID NO:3), LamB3 (Fig. 3D, SEQ ID
NO:4) and LamB4 (Fig. 3E, SEQ ID NO:5).
Figure 4 depicts the signal sequence processing kinetics of the modified LamB signal sequences, LamB1 and ~IJ13~TITUTE SHEET

WOs2/2~665 PCT/US92/05006 - 7 - ,i?,11111~

LamB3. We cultured strain B~21/DE3 containing the appropriate LamB-hBDNF plasmid. In this strain, the gene encoding T7 RNA polymerase has been inserted in the chromosome and is under the~transcriptional control of the lac promoter (Studier and Moffatt, J. Mol. Biol.
189:113-30). Addition of IPTG to the growing cells for 10 minutes allows synthesis of T7 RNA polymerase. Subsequent addition of rifampicin to 0.2 mg/ml blocks transcription by E. coli RNA polymerase but allows transcription by T7 RNA
polymerase. This results in selective transcription of the LamB - hBDNF gene which is placed immediately downstream from the T7 late ~1.1 promoter and ribosome binding site of rbs2. Cells were pulsed with 35S-methionine for 30 seconds and then chased with an excess of cold methionine. The cultures were sampled at the indicated times after chase -~nd the labelled proteins were analyzed by SDS-15% PAGE and fluorography. Processing was determined by densitometric ~
scanning of the precursor and mature forms of LamB-hBDNF. - ~-Figure S is a schematic representation of pRPN121. -The solid line represents pBR322-derîved DNA sequences with the origin of replication (ORI) and the ~-lactamase gene (Ap) indicated. Distinctive features of the plasmid are indicated by boxed regions with arrowheads indicating the dire~tion o~ transcription or replication (ORI). ac W5 is the promoter. rbs2 indicates ~he T7 ~1.1 promoter and the T7 ~1.1 ribosome binding site wi~h minor nucleotide substitutions from the wild type designed to create -~
convenient restriction sites. LamB2 is the signal sequence. hBDNFmyc is the structural gene.
Figure 6 depicts a protein profile of Fast S-SEPHAROSE0 1 fractionation of hBDNFmyc. W3110 I
/pRPN121 extract after DEAE chroma~ography was ~ractionated as described on a 1.6 cm X 6.5 cm Fast S-SEPHAROSE~ column in 7 M urea, 50 mM histidine, pH 5.0, 1 mM EDTA. Fractions as indicated were analyzed by SDS-15% PAGE and proteins -~

SUBSTITUTE SHEET
' , ~ 10 - 8 -visualized by Coomassie stain. Fractions 26-30 were pooled.
Figure 7 is a summary of the purification procedure.
Strain W3110 I~F/pRPN121 was grown, induced, and extracted - as described above. Pooled column fractions were analyzed by SDS-PAGE on a 15% acrylamide gel and stained with Coomassie Blue. Lane 1, DEAE; lane 2, ~ast S-SEPHAROSEX l;
lane 3, Fast S-SEPHAROSE~ 2; lane 4, C4 reverse phase HPLC.
Molecular weight standards are indicated. i Eigure 8 depicts a dose response curve for purified hBDNFmyc stimulation of E8 chicken embryo DRG neurite outgrowth. The hBDNFmyc activity is compared with recombinant hBDNF p~rified from a Chinese hamster ovary cell line. 8Oth proteins were purified to greater than 95%
by C4 reverse phase HPLC.
Figure 9 depicts a dose response curve for the stimulation of E8 chicken embryo DRG neurite outgrowth by recombinant hBDNF purified from ~. Qli- The hBDNF was purified to greater than 95% by C4 reverse phase HPLC.
Figure 10 is a ~chematic representation of pRPN149.
The solid line represents pBR322-derived DNA sequences with the origin of replication (ORI) and the ~-lactamase gen~
(Ap) indicated; Distinctive features uf the plasmid are indicated by boxed regions with arrowheads indicating the direction of transcription or replication (ORI). ~a W ~ is the promoter. rbs2 indicates the T7 ~1.1 promoter and the T7 ~1.1 ribosome binding site with minor nucleotide substitutions from the wild type designed to create convenient restriction sites. LamB1 is the signal sequence. hBDNF is the structural gene.
Figure ll i8 tha DNA sequence of a portion of the isolated human genomic phage clone 7-2 ancoding human NT-4 (SEQ ID NO:22; ATCC Accession No:75070). The predicted hNT-4 protein encoded by the genomic clone 7-2 is represented by the one-letter symbols for amino acids (SEQ

SUBSTITUTE SHEET

W O 92~2266~ ~ t ~ PC~r/US9Z/05006 _ g _ ID No:23). The boxed region represents the predicted cleavage site of the hNT-4 preprotein. Arrows indicate conserved residues in the presequence. The underlined region (N-R-S) represents a consensus sequence for N-glycosylation. The circled region represents the initiating methionine. The splice acceptor site is located at base pair 461-462 (AG), representing the 3'-end of the intron.
Figure 12 is a schematic representation of pRG91. The solid line represents p8R322-derived DNA sequences with the origin of replication (ORI) and the ~-lactamase gene indicated. Distinctive features of the vector are shown by boxed regions. Lac W 5 is the promoter. rbs2 is the phage ;`~
T7 ~1.1 promoter and the T7 ~1.1 ribosome binding site. -~
LamB2 is the signai sequence. -Plasmid pRG91 (Regeneron Pharmaceuticals) is a pBR322 based vector designated for the expression of recombinant proteins and their secretion into the periplasmic space of `
Escherichia coli. The vector consists of the strong, ;~-regulated, lac W 5 promoter followed by the phage T7 ~1.1 promoter and ribosome binding site inserted between the, EcoRI and NruI restriction sites in p8R322. These control elements direct the expression of the LamB2 signal sequence to which recombinant protein gene sequences may be fused.
The DNA sequences between the unique NruI and PvuII
restriction sites were deleted, resulting in increased plasmid copy number. This plasmid confers ampicillin (Ap) resistance;
Figure 13 is a dose response curve for hNT-4 expressed in E. çQl~. Soluble protein extract from an induced c~llture of strain RFJ26 containing plasmid pRG173 was assayed on E8 dorsal root ganglion explants. The background activity of this assay was 0.2 units.
Figure 14 depicts the effect of hNT-4 on C~T activity.
Treatment of motor neuron enriched cultures with a SUBSTI~UTE SHEET

6~ PCT/US92/05006 2111110 - lO- ~`

partially purified extract from an induced culture of strain RFJ26 containing plasmid pRG173 resulted in a 3.6-fold (at 1:20 dilution) increase in CAT activity after 4~
hours as compared to untreated (C-NT) and buffer (C-buffer) controls. The ~. coli extract was passed through a Sepharose-S column as disclosed infra prior to treatment of motor neuron enriched cultures.
5. ~ETAILED DESCRIPTIO~ OF THE INVENTION
As used herein, the term "neurotrophin" refers to any naturally occurring member of the neurotrophin family.
This includes naturally occurring proteins sharing amino acid sequence homology with any known neurotrophin and conserving the six characteristic cysteine residues. (Some of these proteins may fall structurally into the neurotrophin family, yet may exhibit biological activity other than neurotrophic activity.) The term "neurotrophin"
also refers to engineered neurotrophins whose a~ino acid sequences are derived from or patterned after the naturally occurring neurotrophins. For example, it includes chimeric neurotrophins comprising amino acid sequences from different neurotrophins (e.g., NGF and NT-3) or from the same neurotrophin of different species (e.g., hBNDF and pig BDNF); neurotrophins whose genetic sequences have point substitutions, addition or deletion ~utations;
neurotrophins derived from the pre-pro-sequence (e.g., neurotrophins having the two carboxy-terminal amino acid residues encoded by the codons immediately preceding the stop codon of the native gene ("full length neurotrophin")); and includes fragments of a neurotrophin which exhibit neurotrophic activity (e.g., those whose f irst six amino acids are altered or deleted). These examples are descriptive and not meant to limit the definition.
This invention provides processes for producing and recovering biologically active recombinant neurotrophins.

SUBSTITUTE SHEET -~ `

W092~22665 2 1111 1~ PcT/~S~2/0~006 It further provides the purified, homogen~ous, recombinant neurotrophins made by these processes. A recombinant protein, as used in this specification, is a protein expressed from a recombinant DNA molecule in a host cell -.
transformed.. with it. A recombinant DNA molecule is a :-hybrid molecule comprising DN~ sequences from different sources that have been joined together. Sambrook et al.
(19 9, Molecular Cloning: A Laboratory Manuel, Cold Spring ~:
Harbor Laboratory Prec~, Cold Spring Harbor) describes many conventional techniques for recombinant DNA technology.
According to this invention, producing recombinant neurotrophins involves culturing a non-animal host cell transformed with a recombinant DNA molecule having a~
expression control sequence operatively linked to a DNA
sequence encoding a neurotrophin. An expression control ~.
sequence is operatively linked to a DNA sequence encoding a :~.
polypeptide when the expression control sequence directs and promotes the transcription and translation of the DNA
s~.quence. Culturing a transformed host cell involves in~ubating the cell in culture conditions appropriate for . the growth of the cell and the expression of the DNA
sequence.
DNA sequences encoding neurotrophins are available from everal sources. The literature discloses DNA
sequen~es for the four known human neurotrophins (i,e., hNGF (Gray et al., EP 0 1~1 33~, l9B4~, hBDN~ (Hyman et al~, WO 91/03568), hNT-3 ~Hohn et al., WO 91/03569) and Xenop~s NT-4 (Hallbook, et al., 19g1, Neuron 6:845 858) and ~or many non-human neurotxophins. Preferably, one refers to these sequences for direction to construct oligonucleotide primers suitable for PCR ampli~ication.
Genomic libraries are suitable sources of DNA for PCR
templates~ Also useful are cDNA libraries of cells known to express neurotrophin mRNA. For example, cDNA libraries of human fetal brain mRNA contain sequences for hBDNF. One SUBSTITUTE SHEET

W092/2266~ PCT/US92/0~006 Zl lll 10 - 12 -may also screen cDNA and genomic libraries using any of the methods known to the art to identify and isolate DNA
sequences encoding neurotrophins. Alternatively, one may construct synthetic or semi-synthetic genes using conventional DNA synthesizers. Undoubtedly, researchers will discover DNA sequences encoding new neurotrophins.
The methods of this invention will be useful for producing and recovering these neurotrophins, as well.
In specific, the invention relates generally to production of NT-4 or a derivative or fragment thereof by growing a recombinant bacterium containing a nucleic acid encoding NT-4 or derivative or fragment under conditions such that the NT-4 or derivative or fragment thereof is expressed by the bacterium, and recovering the produced NT-4 or NT-4 derivative or fragmen'. In a preferred embodiment, the NT-4 is human NT-4. In another embodiment, .
an NT-4 derivative which is a chimeric or fusion protein is produced. In a most preferred embodiment, the produced NT-4, or NT-4 derivative or fragment is biologically active, i.e., capable of exhibiting one or more of the known functional activities of NT-4, as assayed by any methods known in the art or taught herein ~e.g., n vitro assays of the ~ability to promote outgrowth in E8 DRG
explants, th~ ability to stimulate CAT activity in purified motor neuron cultures; see Section 9, in~a).
In a specific embodiment of the invention, sequences encoding human NT-4 are expressed in an E. coli expression system and a purification scheme as disclosed in~ra is used to produce useful amounts of human NT-4. The nucleic acid encoding human NT-4 which is thus expressed can be that contained in nucleic acid pRG1~3 (ATCC Accession Number 75131) or HG7-2 (ATCC Accession Number 750~0) or shown in -Figure 11 (SEQ ID NO:22), or isolated by any methods known in the art, or as follows: Mixtures of 5' and 3' oligonucleotides representing all possible codons SUBSTITUTE SHEET `

W092/22665 t~ 0 PCT~US92/0~006 corresponding to known NT-4 sequences or to conserved amino acid sequences from known neurotrophins are utilized as primers in the polymerase chain reaction (PCR). Primary and secondary PCR amplification reactions of human (or other mammalian) cDNA or genomic libraries result in the -~
isolation of a PCR product that can be utilized as 32P-labelled probes to isolate a full length cDNA or genomic clone encoding NT-4. The term "human neurotrophin-~", as used herein should be understood as meaning any human homologue of the Xenopus NT-4 (Hallbook, et al., l99:L, Neuron 6:845-858), including a distinct yet homologous te.g., at least about seventy percent homology) ~:
neurotrophin molecule.
The literature discloses a variety of expression control sequences useful for expressing DNA sequences in transformed non-animal hosts. These include, among others, in bacteria, the lac system, the trp system, the ~a~
system, the ~RC system, the lambda PL promoter, the T7 late promoters, and the control regions of the fd coat protein;
and in yeast, the phosphoglycerate kinase promoter, the . Gal 4 promoter, the His promoter, the alcohol dehydrogenase promoter, the alkaline phosphatase prvmoter and the ~-mating fa~or promoter. Controllable expression control sequences are preferable and, a~ong these, a temperature inducible lambda PL pr~moter/ the lac W5 promoter and the T7 ~1.1 promoter are most preferable for expression in E. coli.
-The literature also discloses a variety of expressionvector/host systems suitable for bacterial and fungal hosts, including plasmids, bactPriophages, cosmids and derivatives of them. Examples of these systems are, for bacteria, col F1, pCR1, pBR322, pMB3, RP4, phage lambda, M13 and filamentous viruses; and, for yeast, 2~. Preferred plasmids in bacteria are stable in the host cell and present at medium copy numbers of 20-200 copies per cell.

SUE~STITUTE SHEET

W092/22665 PCT/US92/0~006 ~A 1 ~. 1 1 1 We used plasmids derived from pBR322, such as those described by Panayotatos (1987, Engineering an Efficient Expression System, in: Plasmids-A Practical Approach, ed.
Herdy, K., IRL Press, Oxford/Washington, D.C.). In particular, we prefer plasmids of the RPN clacs, developed by Regeneron Pharmaceuticals, Inc. and described more fully below.
The art is also familiar with many non-animal hosts useful to express heterologous proteins, including ~. coli, Bacillus, Streptomyces, Saccharomyces and Pichia pa~toris.
We prefer ~- ~Qli-Cultùring transformed host cells results in theexpression of neurotrophins. We have found that neurotrophins expressed in bacteria are subject to degradation by intracellular proteases, especially those induced as part of the "heat shock" response to foreign proteins (Goff et al., 1984, Proc. Natl. Acad. Sci. UcA
~1:6647-6651). Therefore, we prefer to use protease-deficient mutants as host cells.
The expression of the heat shock gene~ is regulated ~y heat shock regulatory genes, such as the htpR gene of E. coli. Mutants of the HtR gene are deficient in expression of heat shock protease gene , as well as other genes that contribute ~o ~he heat shock response. We have found that expressing recombinan~ neurotrophins in heat shock regulatory gene mutants, especially htpR mutants, signif icantly improves yield. tpR lon- double mutants are particularly useful. We prefer the ~. oli strain LC137, an htpR~ 1Q~ mutant. This strain is available from Prof.
Alfred Goldberg, Harvard University. U.S. patent 4,758,512 (Goldberg et al.) describes other suitable strains.
Another preferred E . .cQl~ strain is RFJ26.
Neurotrophins are toxic to bacterial cells that express them. Therefore, in order to maximize yield, we prefer to induce neurotrophin expression only in dense SUBSTITUTE SHEET

W092/22~6~ 0 PCT/US92/0~006 bacterial cultures, for example, cultures in late log phase. We have used an inducible system based on the lambda PL promoter and the cI857 temperature sensitive repressor. We typically induce production of neurotrophins by shifting growth temperature to 42C for 30 minutes and continuing incubation for 3-20 hours at 38O-42OC. Inducing expression for more than 16 hours in actively growing cells eventually cau~es cell death.
The inability of E. coli to accumulate neurotrophins may be the result of one or more properties of the neurotrophin gene or protein. The structure of the neurotrophin mRNA, particularly the structure proximal to the translation start point, may prevent efficient translation. ~lternatively, the neurotrophin may prevent its own synthesis by interacting directly with its mRNA, or it may interact directly or indirectly with some component of the DNA replication/transcription/translation machinery of E. ~Qli-Accordinq to another em~odiment of our invention, wesecrete the neurotrophin into the periplasmic space of E. Çgli rather than express it intracellularly. We accomplish this by fusing a signal sequence to a mature neurotrophin. A signal sequence gene fused to the 5' end of the neurotrophin gene may provide a nucleotide sequence proximal to the translation startpoint that is more conducive to efficient translation, thus resulting in higher levels of neurotrophin accumulation. In addition, seguestering the neurotrophin in the periplasmic space prevents it from interfering with any cytosolic component necessary for protein synthesis. It also protects it from attack by cyto~olic proteases. It is also possible that the secretion of a mature neurotrophin into the periplasmic space may provide an environment more conducive to the proper folding of the protein.

SUBSTITUTE SHEET

W092/2266~ ~, PCT/US92/05006 2~ - 16 -A signal sequence is provided by constructing a recombinant DNA molecule in which the DNA sequence encoding the neurotrophin comprises, from 5' to 3', a fused gene encoding a signal or leader sequence appropriate to the host cell which is in-frame with a DNA sequence for the neurotrophin. The literature describes several signal sequences useful in such constructions. For example, LamB, OmpA and PhoA are useful in E. S91i- (Denèfle et al., 1985, Gene 85:~9g-510; Wong et al., 1988, Gene 68:193-203). We prefer LamB and, in particular, modified LamB signal sequences that improve the translational efficiency of the LamB mRNA. We constructed genes for modified LamB signal sequences in the following manner. We made degenerate substitutions to the third nucleotide of several codons of LamB, replacing G or C with A or T. These substitutions do not change the amino acid sequence of the LamB signal peptide, but do decreace the potential number of hydrogen bonds in any secondary structure. This reduces the stability of possible secondary structures involving this region of LamB mRNA. We also introduced co3On changes based on ~odo~ usage models, to more nearly approximate codons used most frequently by E. coli.
We also modified the LamB signal saquence to improve efficiency of processing of the LamB precursor protein into mature protein. Native LamB has a hydrophobic core of 10 amino acid residues. Mutational analysis of several E. ~Qli signal sequences suggests that the length of the hydrophobic core region can have a strong effect on signal sequence activity. We have found that increasing the length of the hydrophobic region by the addition of up to ten hydrophobic amino acid residues improves the ef~iciency of processing LamB fusion precursor polypeptides. Fewer than six is preferable and four is most preferable. The choice of hydrophobic amino acids added is not critical, nor is the precise location at which they are added to the SUBS~ITUTE SHEET

W092/2266~ 0 Pcr/uss2/o~oo6 hydrophobic core region. However, we prefer to add the tetra-peptide Leu-Ala-Val~Leu ("LAVL") (SEQ ID NO:6) at a convenient restriction site near the N-terminal end of the hydrophobic region. We describe particular genes for modified LamB signal sequences in Example 7.
In accordance with our invention, the recombinant neurotrophins are released from the culture of host cells by harvesting the cells, lysing them, centrifuging the lysate and collecting the lysate pellet. One typically harvests the cells by centrifugation. To inhibit degradation of the neurotrophin after harvesting the cells, one preferably resuspends them in 50 mM EDTA. Then we either use the cells directly or freeze them for later use.
The art is familiar with many techniques to lyse cells including enzymatic (e.g., lysozyme), chemical (e.g., alkali, SDS) and mechanical (e.g., French press, hydrodynamic shear). Mechanical means are preferred because there is less risk of harm to the neurotrophins.
We prefer to lyse the cells by passing them through a French press or a STANSTED~ cell disrupter at 10,000 psi.
We obtain the lysate pellet by centrifugation, at about ~`
15,000 x g.
This invention further provides processes ~or recovering t~e recombinant neurotrophins produced in cell cultures. These processes o~ercome the problems associated with conventional methods of recovering recombinant proteins that have been applied to neurotrophins.
Native neurotrophins are soluble in neutral buffers.
However, recombinant neurotrophins from E. ~1~ behave as insoluble proteins. Recombinant neurotrophins have been detected in the cytosolic fraction and, when exported, have been recovered from the periplasmic space using standard techniques such as osmotic shock, spheroplasting, or -freeze-thaw (Bochner et al., U.S patent 4,680,262).

SUBSTITVTE SHEET

W092~22665 ,~ ~ PCT/US92/0~006 2 ~ 10 - 18 -Howe~er, they are isolable only as a small fraction of the total neurotrophins in the cell.
Many mammalian proteins expressed in bacteria are insoluble. Produced in a foreign ionic environment, they do not fold correctly and expose normally hidden hydrophobic regions. Consequently, the proteins form aggregates and precipitate as inclusion bodies. Thei literature suggests that the cysteine residues of these proteins are at least partially reduced or mismatched (Tsuji et al., 1987, Biochemiætry 26:3129-3134).
The literature describes standard techniques for purifying recombinant proteins from inclusion bodies (see, ;~
e.g., Builder et al., U.S. patent 4,620,948; Hershenson et al., U.S. patent 4,961,969; and Hung et al., U.S patent ;~
4,734,362). These techniques involve dissolving the protein in a solution comprising a strong denaturant and a reducing agent. After exchanging the strong denaturant for a weak denaturant, the proteins are renatured in a neutral solution and oxidized to form the correct disulfide bonds.
The majority of the recombinant neiurotrophin produced ~-by these methods is biologically inactive. We believe that ~-this is due, in part, to the complexity of the neurotrophins' tertiary structure. After denaturing the protein and reducing the sulfhydryl groups, the polypeptide does not refold into the proper configNration for the correct disulfide bonding. Furthermore, our results suggest that recombinan~ neurotrophins are present in aggregates tha~ ar~ not typical inclusion bodies.
We have discovered a process to recover biologically active recombinant neurotrophins from the insoluble host cell fraction. Essentially, the process involves solubilizing the neurotrophin in a strong denaturing agent while maintaining the correct oxidation state of the protein by avoiding the use of reducing agents. Reducing the disulfide bonds during purification destroys the W092~2266S ~ PCT/US92/OS006 ., .
activity of purified neurotrophin polypeptides. Because the literature (e.g., Tsuji et al. 1987, Biochemistry 26:3129-3134) teaches that insoluble recombinant proteins in inclusion bodies should have incorrectly formed or reduced disulfide bonds, this is an unexpected result.
To further recover the neurotrophins, the strong denaturing agent is replaced with a weaker one. We have also discovered, however, that the addition of a basic amino acid, such as histidine, or its equivalent, helps keep the neurotrophin soluble during the renaturation process that accompanies the removal of the weak denaturant (Prior et al., lg90, WO 90/06764).
These discoveries suggest to us a new model for the ;~
behavior of recombinant neurotrophins. We believe that the ma~ority of the recombinant neurotrophin polypeptides produced by the host cell fold properly and form the ~-correct disulfide bonds. Furthermore, since neurotrophins have a high pH and are positively charged at neutral pH, we believe that they form associations with negatively charged molecules in the cell, for example DNA, RNA and other pro~eins. Consequently they become insoluble at neutral pH. Basic amino acids help to dissociate them.
More specifically, our process for recovering recombinant neurotrophins produced ~y a host cell culture co~prises the step of denaturing the neurotrophin by diæsolving it in a solution ~omprising a strong denaturing agent which solution is also essentially free of reducing agents. Denaturing agents as used herein refer to compounds which, in aqueous solution, reversibly unfold dissolved proteins by at least partially eliminating tertiary and secondary structure throu~h the disruption of hydrogen bonds or alteration of the thermodynamic surroundings of the protein. Strong denaturing æolutions include guanidinium salts ~e.g., guanidinium hydrochloride) and alkali metal thiocyanates (e.g., sodium thiocyanate) at SUBSTITUTE SH~ET i ;.

2~ A~IG 1993 ~TAJS 9 2 l 0 5 0 0 ~'111110 concentrations of 4 M - 9 M, and urea at 7 M - 9 M. The preferred denaturing solutions are 7 M - 9 M guanidinium HCl. Preferred conditions are pH 7.0 - pH 9.0 and room temperature. Most preferred is 8 M guanidinium HCl, pH
8Ø
One must also maintain the correct oxidation state of the sulfur atoms in the cysteine residues during this step or risk losing the ability to recover any biologically active neurotrophins. Therefore, the solution must be essentially free of reducing agents. A solution essentially free of reducing agents is one in which a protein's disulfide bonds are maintained. Addition o~ even small amounts of disulfide reducing agents, such as ~-mercaptoethanol or dithiothreitol, during the practice of this invention will destroy the activity of the purified neu-otrophin. `
Recombinant neurotrophins apparently are subject to degradation by metalloproteinase~. Thexefore, preferably, one recovers neurotrophins in solutions comprising metalloproteinase inhibitors. We prefer heavy metal chelators, such as EDTA. The concentration ~f EDTA may range from a minimum of at least 1 mM to a maximum of about 200 mM. Concentrations of 5 mM - 80 m~ are preferable a~d 50 mN is most preferable. After chelated heavy metal ions hav~ been dialyzed from the solution, EDT~ may be reduced or ~l~minated~ Langley et al., 1990, EP 0 398 753, describes`peptides useful as metalloproteinase inhibitors.
The recovery process may also comprise one or more of the following steps. After solubilizing the neurotrophin, one exchanges the strong denaturing a~ent in the solution for a weak denaturing agent. Weak denaturing solutions include ur~a at 4 M - 9 M and, preferably, 6 M - 8 M, pH

7.0 - pH 9.0, at room temperature. The most preferable weak denaturing solution is 7 M urea, 50 mM Tris-HCl, lO mM
NaCl, 5 mM EDTA, pH 8Ø Dialysis is the preferred method W092t2266~ 0 PCT/US92/0~006 of exchange. If the strong denaturing agent is 7.0 M - g.o M urea, the weak denaturing agent of this step is preferably urea of lower concentration.
In order to renature the neurotrophin, one removes the weak denatu~ing aqent from the solution. Dialysis or diafiltration against a non-denaturing solution is the preferred method of removal. We prefer 0 M to 1 M NaCl as :
a non-denaturing solution.
Another step in the recovery process is purifying the recombinant neurotrophin from other contaminants in the solution. Any of the typical protein isolation techniques known to the art may be used. We prefer a three step isolation involving cation exchange on S-SEPHAROSE~, anion exchange on DEAE-SEPHAROSE0 and reverse phase high pressure ` -liquid chromatography (HPLC). The isolation step can begin at any stage after solubilization of the neurotrophin and may continue through the denaturing agent exchange and removal steps. Preferably, we begin purification at the urea phase, because this agent does not interfere with ion exchange chromatography and because partial purification made the neurotrophin easier to dissolve in the non-denaturing.solution.
We have found that unless the neurotrophin is relatively purified it will precipitate out of the solution when one removes the weak denaturing agent. Solubility depends upon degree of purity of the neurotrophin as well as the character of the other contaminants in solution.
For example, negatively charged molecules such as DNA will interfere with solubility of even very pure neurotrophin.
Therefore, we maintain the æolubility of the neurotrophin in the non-denaturing solution ~y adjusting the solution to comprise a basic amino acid or an equivalent, such as indole acetic acid. The concentration of this species -should be effective to maintain the neurotrophin in solution. Solutions of basic amino acids include SUBSTITUTE SHEET

W092/22665 i PCT/US92/0~006 ~ 22 -histidine, lysine and arginine or their salts at concentrations above lO mM. Histidine in concentrations of 20 mM - 500 mM is useful. We most prefer histidine in concentrations of 20 mM - lO0 mM. If the neurotrophin in the denaturing solution has been sufficiently purified before removal of the denaturant, this step may be eliminated.
Results indicate that our method produces uniform neurotrophin molecules, consistently having the same N-terminal amino acid. In contrast we have found that expression of naurotrophins in the mammalian CHO cell system produces a mixture of neurotrophin molecules with varying N-terminal amino acids. Therefore, the recombinant neurotrophins produced by our process appear to be unique.
The processes of this invention are also useful for recovering other proteins having biochemical properties similar to the neurotrophins. That is, the processes are useful for recovering proteins that fold correctly in bacteria and form the proper disulfide bonds, but, once denatured and reduced in conventional recovery techniques, are very difficult to renature with proper disulfide bonds.
This includes proteins with pH greater than about 9.0 and having at least two disulfide bonds. (Candidate molecules include secretory leukocyte protease inhibitor (Miller et al., 19~9, J. Bacteriology 1~1:2166-21?2) and full length recombinant CD4 (Fisher et al., 1989 WO 89101940) .) - As described in an example section inf~a, the present invention discloses the expression of biologically active human neurotrophin-4. The human NT-4 DNA sequence was subcloned into the DNA plasmid vector pRG91, resulting in ~`
pRG173. This bNT-4 containing plasmid was transformed into E. coli strain RFJ26, and methods described in the instant specification were utilized to recover biologically active `~
N~-4 from the culture system. However, applicants are not ~`
to be limited to such a specific embodiment. For example, SUBSTITUTE SHEET ` `
~. .

W092/2266~ ~) t I ~ I ~ PCT/US92/0~006 any nucleic acid sequence substantially homologous to the region of HG7-2 encoding human NT-4 can be utilized to construct any number of DNA plasmid expression vectors as described throughout the specification or known to the skilled artisan, which in turn can be utilized to transform any number of E. coli bacterial strains in order to produce useful amounts of biologically active NT-4.
In order that this invention may be better understood, the following examples are set forth. These examples are for the purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

6. EXAMPLE: PRODUCTION AND RECOVERY OF
RECOMBINANT hNGF F~OM E. CO~I
A DNA sequence carrying the mature human NGF (hNGF) gene was amplified from human genomic DNA using known human NGF sequences (Nancy Ip; Regeneron Pharmaceuticals, Inc.) by PCR amplification. We used the oligo-deoxynucleotide primers PAN-20 (5'-AAGCGGTCGA CATCTOATCC AATCTTCCAT
AGAGGTGAAT TCTCAGTA-3') ~SEQ ID NO:7~ and EVD-3 (5'-GGCAGGCGGÇ CGTCATCTCA CAGCCTTCCT GCTG-3') (SEQ ID
NO:8). These primers were designed to incorporate a SalI
restriction site at the 5' end and an aqI restriction site at the 3' end of the human NGF gene. In order to incorporate a ~lI site at the 5' end with PAN-20, it was necessary to change the second amino acid from the amino terminal sequence of the mature hNGF protei.n from serine to the structurally similar amino acid threonine. In view of the limited sequence identity of the three known neurotrophins in t~eir eight N-terminal amino acids, such conservative amino acid changes should not affect activity.
Furthermore, the identity of the amino acid residue in this position is not preserved in NGF molecule~ from other species ~Ibanez et al., 1990, EMBO J. 9:1477-1483). In addition, PAN20 was designed so as to lower the ~+C content SUBSTITUTE SHEET

W092/2266~ PCT/US92/05006 , 2 ~ 1 - 24 -of this region of the NGF gene in a manner that does not affect the sequence of the resulting protein.
rhe amplified DNA fragmen~ was digested with the restriction endonucleases SalI and EaaI and ligated between the SalI and ~I sites of p~PN50, (Regeneron Pharmaceuticals, Inc.), resulting in plasmid pRPN102.
pRPN50 is derived from expression plasmid pNKS97 (Panayotatos, 1987, Engineering an Efficient Expression System, In: Plasmids-A Practical Approach, ed. Herdy~ K., IR~ Press, Oxford/Washington, D.C.) by the insertion of the lambda PL promoter at the promotor insertion site~
The cI857 lambda PL repressor gene was incorporated into pRPN 02 to facilitate repression at 30C and to allow derepression by heat shock at 42C. This DNA sequence was ~`
PCR amplified using the primers EYD-26 (5'-CCATTATCGC
GACCAGAGGT-3') (SEQ ID NO:9~ and EVD-27 (5'-TCTTGCTCGC
GAGTTATCAG CT~T~CG-3')(SEQ ID NO:10) that generate a NruI `
restriction site at each end. This 821 bp fragment extends 70 bp upstream from the cI857 coding sequence in a region that contains the PRM constitutive promoter as well as 38 bp beyond the cI857 termination codon. The PCR amplified -~
cI857 fragment was digested with ~I and ligated into the NruI site of pRPNl02. Candidates from this ligation were screened to find a plasmid with the cI857 insert that had the same transcriptional ~irection as the h~GF gene, resulting in plasmid pRPN133 (Fig. ~). Plasmid pRPN133 has been deposited with the ATCC and has been assigned Accession No: 75029.
We transformed the E. ~Q~ R~ Rts 1Q~- mutant strain, LC137, with RPN133 to yield pRPN133/LC137.
In a 2 liter flask, 400 mL of LB medium supplemented with 100 mg/L ampicillin was inoculated with pRPN133/LC137 and incubated at 28C with shaking. At late logarithmic -`
growth phase ~OD5~ = 1.0 - 2.0 under these conditions), hNGF -synthesis was induced by adding to the overnight culture SUBSTITUTE SHEET `:

400 mL of LB media preheated to 55C and continuin~
incubation with shaking at 42C for 5 Aours.
Cells were harvested by centrifugation and the cell pellets stored at -20C. Cell pellets (1.0 - 2.5 g) were thawed, resuspended in 20.0 mL buffer A (100 mM Tris-HCl, 50 mM EDTA, pH 8.0), passed through a STANSTED~ cell disrupter at 10,000 psi, and centrifuged at 23,000 x g for 15 minutes at 4~C. The pellet was washed twice with 10.0 mL of 2 M guanidinium-HCl, 5 mM EDTA, pH 8Ø
To solubilize the recombinant hNGF, we resuspended the pellet in 40 mL of 8 M guanidinium-HCl, 5 mM EDTA, pH 8.o using a Potter homogenizer. Then we centrifu~ed the suspension at 23,000 x g for 15 minutes.
The supernatant was dialyzed against 2 liters buffer B
t7 M urea, 100 mM histidine, 0.1 mM EDTA, pH 6.0) with two buffer chanqes at 25C for 20 hours. The dialysate was centrifuged at 23,000 x g for 15 minutes at 4C.
The supernatant was applied to an S-SEPHAROSE~ column (2.5 cm x 6.0 cm d x h) equilibrated in buffer B and washed to baseline absorbance at 280 nm with the same buffer.
Proteins were eluted with a gradient of 300 mL of 0.0 M -1.0 M NaCl in buffer B. Fractions containing hNGF were pooled.
The pooled fractions were dialysed against 100-fold exce~s volume buffer C (7 M urea, 50 mM Tris-HC1, 0.1 mM
EDTA, pH 8~5) at 25C for 20 hours.
The dialy~ate was applied to a ~EAE-SEPHAROSE column t2.5 x 7.5 cm d x h) equilibrated in buffer C at a flow rate of ~.O mL per minute. The flow-throu~h fractions containing the hNGF were pooled and dialyzed twice against 40 volumes of 100 mM histidine, 0.1 mM EDTA, pH 6.0 at 4C
overnight. The dialysate was centrifuged at 23,000 x g for 15 minutes at 4C. The dialysate was then applied to a TOYOPEARL CM 6505~ (weak cation exchange) column (1.0 x 5.0 cm) equilibrated with 100 mM histidine, 0.~ mM EDTA, pH 6.0 W092/22665 PCTJUS92J0~006 ~ 10 - 26 -and eluted with a 0.0 M to 1.0 M NaCl gradient. Fractions containing the hNGF were pooled and filtered through MILLIPORE GV~ filter and stored at 4C. The amino terminal sequence of the purified protein was confirmed by direct sequencing.
The biological activity of the purified protein was tested for neurite outgrowth in E8 explanted and dissociated dorsal root ganglia (Fig. 2A and 2B) (Lindsay et al., 1985, Dev. Biol. 112:319-328). By this criteria the recombinant hNGF purified from E. coli by this method was found to be as active as NGF purified from mouse salivary gland.

7. EXAMPLE: PRODUCTION AND RECOVERY OF
RECOMBINANT hBD~F and hBDNFmyc FROM E. COLI
7.1. S~N~ SEQUENCES BASED ON L~MB
We constructed signal sequences based on Lam8 to `~
facilitate both the synthesis and secretion of neurotrophins in E. coli. The LamB signal 6equence (Fig.
3A, SEQ ID NO:1) is a naturally occ~rring E. coli signal sequence which was selected for the construction of a series of se~retion vectors based on the pRPN series of expression vectors developed at ~egeneron Pharmaceuticals, Inc. Secretion vectors were constructed by cloning synthetic DNA fragments encoding the LamB signal sequence `
into pRPNO9 or pRPN16. These plasmids derive from expression vector pNXS97 (Panayotatos, 1987, Engineering an Efficient Expression System, In: Plasmids-A Practical Approach, ed. Herdy, K., IRL Press, Oxford/Washington, D.C.) into which have been inserted a lac W 5 promoter.
LamB was inserted into the structural gene insert site such that expression of Lam8 is under control of the lac W5 or T7 ~1.1 promoter.
The synthetic DNA fragment, LamB1 (Fig. 3B, SEQ ID
NO:2), encodes 2S amino acids identical to the wildtype SUBSTITUTE SHEET

W092/2266~ O PCT/US92/05006 LamB signal sequence. However, we altered the nucleotide sequence relative to the wild type nucleotide ~equence for the purpose of constructing unique restriction enzyme sites and for the maximization of translation efficiency. LamBl also has seven degenerate nucleotide substitutions replacing C or G with A or T. This reduces the stability of possible secondary structure of mRNA. LamBl also has several new restriction sites.
We also created LamB2 (Fig. 3C, SEQ ID NO:3) as a modification of Lam81. To make Lam~2 we made nucleotide changes to the 3' end of LamBl by PCR amplification. These changes introduced an EagI restriction site which facilitates the cloning of blun~ end DNA fragments.
The fusion of mature hBDNF to LamB1 results in efficient synthesis of the fusion protein in E. coli. The authenticity of the synthesized product was confirmed by selective synthesis in a DNA-dependent coupled transcription-translation cell-free protein synthesis system, by the selective synthesis of the product using a T7 RNA polymerase expression system in E. ~ , and by the synthesis of the product with a C-terminal myc tag allowing for identification of the chimera with a myc-specific monoclonal antibody. Either one of these fusion proteins synthesized in ~. coli was processed to mature h3DNF as evidenced by i~s mobility on SDS-PAGE. The level of expression obtained with LamB1 or LamB2 results in accumulation of hBDNF from about 1% to 10% of total cell protein.
We modified LamB1 to increase the efficiency of procassing. Four amino acids (Leu-Ala-Val-Leu, or LAVL) were inserted into the ~heI site of LamBl to yield LamB3 (Fig. 3D, 5EQ ID NO:4). The same insertion was made in LamB2 to yield LamB4 (Fig. 3E, SEQ ID NO:5). This insertion results in extension of the hydrophobic core region of LamBl and Lam~2 from 10 to 14 amino acids. It SUBSTITUTE SHEET

W092/2266~ , PCT/USg2/0~006 ZlllllO - 28 -results in a 5-fold increase in the rate of processing of the hBDNF fusion protein into mature hBDNF (Fig. 4). :
LamB3- and Lam~4-hBDNF molecules that are exported into the periplasm are processed into mature hBDNF more rapidly than wild type LamB fusions. However, fewer molecules are exported~ so the net amount of mature hBDNF
in this case is not increased. In any case the increased translocation efficiency of LamB3 and LamB4 should result in improved yields of other proteins.
The LamB1 signal sequence fragment was constructed as two complementary synthetic oligonucleotides tLamB80, 5'-ATGATCACAC TGCGTAAGCT TCCGCCTAGCT GTAGCAGTAG CAGCAGGTGT
AATGTCTGCA CAGGCCATGG CCCGGGATCC-3' (SEQ ID NO:11) and LamB88, 5'-CTAGGGATCC CGGGCCATGG CCTGTGCAGA CATTACACCT ~-GCTGCTACTG CTACAGCTAG CGGAAGCTTA CGCAGTGTGA TCATCATG-3'(SEQ `~
ID NO:12)), designed so as to generate upon annealing `
protruding ends corresponding to those of the SpeI and SphI
restriction sites. This DNA fragment was ligat d into the SDhI and ~peI restriction sites of pRPN16 (Regeneron ` Pharmaceuticals, Inc.) resulting in plasmid pRPN52.
This plasmid was subsequently modified, for the purpose of creating additional restriction sites, by the insertion o~ a synthetic DNA fragment, con isting of the annealed product of the complementary LamB2A, ~S'-CATGGCrAGT CGGCCGAG-3' (SEQ ID NO:13)~ and LamB2B
(5'-GATCCTCGGC CGAC~GGC-3' (SEQ ID NO:14)) oligonucleotides, between the unique NcoI and ~E~I
restriction sites in the LamB1 signal sequence in pRPN52.
The resulting plas~id, pRPN88, contains the LamB2 signal sequence with unique restriction sites at the signal peptidase recognition sequence to facilitate fusion of the signal sequence to any other DNA sequence. The LamB2 signal sequence in pRPN88 is under transcriptional control of the lac W5 or the T7 ~1.1 promoter and translational control of the T7 ~1.1 ribosome binding site.

SUBSTITUTE SHEET

W092/22665 ~ 0 PCT/US92/0~006 -- 2g --7.2. PRODUCTION AND RECOVERY
OF RECOMBINANT hBDNFmyc Human BDNFmyc is a protein comprising, from N- to C-terminus, mature hBDNF fused to an antigenic ~'tag." The tag is a peptide having the amino acid sequence EQKLISEEDL
(SEQ ID NO:15~ These ten amino acid residues derive from the human c-myc proto-oncogene. Antibodies recognizing this tag are useful for identifying hBDNFmyc in a sample (Evan et al., Mol. Cell. Biol. 5:3610-3616; see also, S.
Squinto et al., "Assay Systems for Detecting Neurotrophic Activity," U.S. application 07/5321,283, incorporated herein by reference).
To construct a fusion protein comprising the LamB2 signal sequence and the hBDNFmyc protein, the hBDNFmyc DNA
sequonce was PCR amplified from pCDM8-hBDNFmyc (Regeneron Pharmaceuticals, Inc.) using oligonucleotide primers N8-hBDNF (S'-CCCACTCTGA CCCTGCCCGC CGAGGG-3' ~SEQ ID
NO:16)) and C2-hBDNFmyc (5l-GCTATGCGGC C~CT~CAGAT
CCTCCTC-3' (SEQ ID NO:17). The amplified DNA fragment was digested with EaqI and cloned into the ~lI and ~gI sites of the LamB2 sequence in pR*N88, resulting in plasmid pRPN98.
One also can produce a ~N~ sequence encoding hBDNFmyc by fusing a DN~ sequence encoding mature hBDNF to one encoding the ten-amino acid myc ta~. ~NA sequences encoding mature h~DNF are isolated by PCR amplification as dascribed. (Hyman et al., l991, WO 091/01568.) A double stranded DNA sequence encoding the ten-amino acid myc peptide tag may be chemically syn~hesized. For the purposes of this Example, the hybrid hBDNFmyc DNA sequence is then provided with ~lI and Ea~I restriction sites at the 5' and 3' ends, respectively.
It should be noted that there are two ~lI sites in pRPN88 but the site in the pBR322-derived sequences is about 50 times less sensitive to BalI cleavage than the site in the LamB2 signal sequen~e due to dcm methylation SUBSTITUTE SHEET

W092/2~66s PCT/US92/0~006 I1 1110 ~ 30 -(New England Biolabs). In pRPN98 the LamB2 signal sequence is fused to the mature part of the hBDNFmyc protein so that cleavage at the signal peptidase recognition sequence .
should yield hBDNFmyc protein starting at the histidine residue at +1 relative to the pro-protein processing site (Leibrock, 1989, Nature 341:149-152).
The DNA sequences between the unique ~E~I and ~
sites in pRPN98 were deleted to remove sequences that ~-.
control copy number of the plasmid (Twigg and Sherratt, 1980, Nature 283:216-218). The resulting plasmid, pRPNl21 (Fig. 5), has a copy number about 3-fold higher than the parental plasmid. Plasmid pRPN 121 has been deposited with the ATCC and been assigned Accession No: 75028. : :
We transformed ~. coli W3110 IqF- with pRPN121 to produce W3110 Iq~/pRPN121.
In a 2 L flask, 500 ml of LB medium was inoculated -with W3110 Iq~/pRPN121 and grown to late log with shaking ~;
at 37C (OD5~ approximately 1.0), after which lactose was added to a final concentration of 1% (w/v) and the culture aerated overnight at 37C. Cells were harvested by centrifugation, washed once in 0.2 M Tris-KCl, pH 8.0 and the cell pellet frozen at -70C.
The cell pellet ~approximately 5 g) was thawed and resuspended in 20 mL 0.2 M Tris-HC1, pH 8.0, 1 mM CaCl2, and 25 units micrococcal nuclease (Boehringer Mannheim). The cell suspension was passed through a French pr~ssure cell at 8000 psi and then centrifuged at 15,000 rpm for 15 minutes in a SA600 rotor at 4C. The pellet was resuspended in 30 mL of 0.2 M Tris-HCl, pH 8.0, 10 mM EDTA, 2% Triton X-100 and gently rocked at room temperature for one hour then centrifuged at 15,000 rpm for 15 minutes in a SA600~ rotor at 4C. The pellet was washed twice in 20 mL
of 2 M guanidinium-HCl. The pellet was resuspended in 10 mL of 8 M guanidinium-HCl, 10 mN Tris-HCl, pH 8.5, 10 mM

SIIE~TITUTE SHEET

W092/22665 PCT/USg2/05006 1lilO

NaCl, 1 mM EDTA using a Potter homogeni~er. The extract was brought to 20 mL with the same buffer.
The extract was dialyzed overnight at room temperature against 100 X volume 7 M urea, 50 mM Tris-HCl, pH 8.5, 10 mM NaCl, 1 mM EDTA.
The dialysate was applied to a DEAE ZETA-PREP~ disk (Cuno, Inc., Meriden, CT) equilibrated in 7 M urea, so mM
Tris-HCl, pH 8.5, 1 mM EDTA, at a flow rate of 3 mL/minute and washed to baseline with the same bu~fer.
The flow-through fractions were brought to 50 mM
histidine, pH 5.0 then dialyzed overnight at 4C against 100 x volume 7 M urea, 50 mM histidine, pH 5.0, 1 mM EDTA.
The dialysate was applied to a 1.6 cm x 6.5 cm column of S-SEPHAROSE0 equilibrated with 7 M urea, 50 mM histidine, pH 5.0, 1 mM EDTA, at a flow rate of 1 mL/minute, and washed to baseline with the same buffer. Proteins were eluted with a NaCl gradient from 0.0 M - 1.0 M in 200 ml.
Fractions containing hBDNFmyc were collected tFig. 6).
Fractions containing hBDNFmyc were dialyzed against 200 X volume 50 mM histidine, pH 5.0, 50 mM ~aCl, 1 mM
EDTA. The dialysate was applied to a 1.6 cm X 1.5 cm column of S-S~PHAROSE0 equilibrated with 50 mM histidine~ ;
pH 5.0, 50 mM NaCl, 1 mM EDTA, at a flow rate of 1 mL/minute and washed to baseline with the same buffer.
Proteins were eluted with a NaCl gradient from O.O M - 1.0 M in 200 ml. The fr~ctions containing hBDNFmyc were collected and pooled.
The hBDNYmyc in this sample (approximately 85% pure) was applied directly to a 0.45 cm X 5.0 cm C4 reverse phase column (VYDAC~) and eluted with a gradient of 0-67%
acetonitril~ in 0.1% trifluoroacetic acid in 80 mL at a flow rate of 0.75 mL/minute. The peak fraction was greater than 95% pure (Fig. 7).
Analysis of the N-terminal amino acid seguence confirmed the purified protein to be authentic h8DNFmyc SUE3STITUTE SHEET `:

WOg2/2~665 ..:~ PCT/US92/0~006 , 2 ~ 0 ~ 32 -:' with a homogeneous N-terminus (Leibrock, 1989, Nature 341:149-152).
The purified hBDNFmyc was biologically active in promoting neurite outgrowth from dorsal root ganglion (DRG) `~
explants and nodose ganglion explants from E8 chicken ~:
embryos, as well promoting survival and dendritic outgrowth of dissociated neurons from E8 chicken DRG's (Fig. 8).
This activity is comparable to recombinant human BDNF
purified from mammalian cell extracts on DRG explant assays. Material assayed corresponds to Fig. 7, lane 4.

7.3. PRODUCTION AND RECOVERY OF
RECOMBINANT ~DNF
The process described for the production and recovery --of hBDNFmyc was also used to produce and recover recombinant mature full length hBDNF, and yielded protein with similar bioactivity (Fig. 9). The construction of plasmid pRPN149 (Fig. lO), which expresses a LamB1-hBDNF
fusion protein, is analogous to the construction of .-pRPN121. The synthetic LamB1 DNA fragment,~described .:.
above, was cloned into the ~E~I and SpeI restriction sites of pRPNO9 (Regeneron ~harmaceuticals, Inc.) resulting i~
plasmid pRPN31.- The mature hB~NF DNA sequence was PCR
amplified from pCDM8-hBDNF ~Regeneron Pharmaceuticals, Inc.~ using oligonucleotide primers N1-hBDNF (5'-ACTCTGACCC
TGCCCGCCGA GG~GAGCTG-3') (SEQ ID NO:18) and Cl-hBDNF
(5l-GCGCGGATCC CTATCTTCCC CTTTTAATGG TCAATGTAC-3'~ tSEQ ID
NO:19) This DNA fragment was clsned into SmaI and BamHI
sites of pRPN31 resulting in plasmid pRPN34. The 3EHI fragment including the LamBl-hBDNF fusion gene was su~sequently cloned into the ~in~ 3mHI
restrict~on sites of pRPN52 (described above) resulting in plasmid pRPN66. The NruI-Ey~II deletion of pBR322 sequences resulting in higher copy number (described above) was made in p~PN66 resulting in plasmid pRPN149. In this .-~
plasmid, expression of LamB1-hBDNF is under control of SUBSTITUTE SHEET

W092/226~ J 1. t 1110 PcT/US92/05006 lac W5 and the T7 ~1.1 promoter and ribo~ome binding site~
Plasmid pRPN149 has been deposited with the ATCC and has been assigned Accession No: 75027.
We transformed E. coli IqF~3110 with pRP~149. Then we produced and recovered recombinant hBDNF by the procedure described in Example 7.

8. EXAMPLE: PRODUCTION AND RECOVERY OF
_ RECOMBINANT h~T-3 Human NT-3 is produced and recovered by processl~s similar to those we described for hNGF and hBDNF.
A DNA sequence encoding mature full length hNT-:3 is PCR amplified from a cDNA library from h~man brain cells (Hohn et al., 1991, WO 91/03569, incorporated herein by reference). The oligonucleotide primers EVD-45 (5'-CCTATGCAGA GCATAAGAGT CACCGAGGA-3') (SEQ ID NO:20~ and EVD-7 (5'-GTAAGGGCGG CCGAAGTTTA ATAAATAAAG GTC-3') (SEQ ID
NO:21) are u~ed. These prim~rs are derived f~om the DNA
sequence for rat NT-3 (Maisonpierre et al., Scie~ce 24? :1446-1451). The sense primer is nearly'identical to the human NT-3 sequence. The antisense primer hybridizes approximately one hundred base pairs clownstream of the termination codon of the human gene.
This DNA fragment has a C terminal Ea~I restriction site suitable for insertion into ~31I and E~qI sites of pRPN88, where it would replace the h~DNF ~NA ~equence. The resulting plasmid is used to tran~form E. coli IqF~3110.
Then recombinant hNT-3 is produced and recovered as in Example 7. We expect recombinant hNTo3 purified from coli ~o exhibit n~urotrophin activity similar to that descri~ed by Hohn et a.l., 1991, ~Wo 91/03569~.

SUBSTITUT SHEET

W092/2266~ PCT/US92/050~6 . - 34 -9. EXAMPLE: PRODUCTION AND RECOVERY OF
RECOMBINANT hNT-4 FROM E. CQLI
9.1. CONSTRyCTION OF pRG173 The DNA sequence encoding the putative mature region of th~ human NT-4 (hNT-4) gene was amplified by PCR from NT-4 HG7-2 DNA, using the N1-NT4 (5'-CCGGGGTCTCTGAAACTGCACCAGCGAGTCG-3') ~SEQ ID NO:23] and C1-NT4 (5'-GGTGCAGTTTCAGAGACCCCCATACGCCGGCTGCGGTTGGC-3') ~SEQ ID NO:24] oligonucleotides as primers. The Cl-NT4 oligonucleotide generates an EagI restriction site 3' to the NT-4 gene. The PCR generated fragment was digested with EagI and cloned into MscI-EagI digested p~G9~. The resulting plasmid, pRG173, consisted of the LamB signal sequence fused to the mature region of hNT-4 (the glycine at amino acid residue 81 in the HG7-2 translat~d sequence) under the transcriptional control of the lac W 5 and T7 ~1.1 promoters and the translational control of the T7 ~1.1 ribosome binding site. This plasmid lso possessed the ropl deletion which increa es plasmid copy number. The construction was confirmed by restriction enzyme analysis of purified plasmid DNA, ~NA sequence analysis of the purified plasmid, n VltrO synthesis of a protein of the' approximate size estimated to be encocled by p~l73, and ln ViVQ synthesis of a protein of the appropriate size estimated to be encoded by pRG173 and poss~ssing neurite outgrowth stimulating activity (see discussion, sup~a), and having the appropriate N-terminus as determined by amino acid sequencing ~GVSETAPAE tSEQ ID NO:25~).

9.2. PURIFICATION OF HUM~N NT~4 ~XPRESSED
IN ~ ~O.~I-EEI~L~G173 __ A 5 ml culture of pRG173 transfo~med in E. oli strain RFJ26 was grown overnight in LB medium, supplemented with 100 mg/L ampicillin at 37C with aeration. The overnight culture was diluted into 500 ml LB and grown to OD5~ = 5.3 -SUBSTITUTE SffEET

W092~2266~ PCT/US92/0~006 ~ 2tllllO

at which time the expression of hNT-4 was induced by the addition of lactose to 1% (w/v) and the culture was grown overnight with aerat~ion. Cells were then collected by centrifugation and frozen at 70C. The cell pellet (7.2 grams) was thawed and resuspended in 73 ml of 200 mM
Tris-HCl, pH 8.0, 50 mM EDTA and lysed by 3 sequential passes through the STANSTED~ cell disrupter. The lysate was then centrifuged at 26,000 x g for 10 minutes, yielding 83 ml of supernatant containing the soluble fraction. The soluble fraction was dialyzed against 50 mM Tris-HCl, pH
8.5 at 4C for 5 hrs then diluted 10-fold with 20 mM MES, -pH 6.0 and loaded on a Fast S-Sepharose column equilibrated with 20 mM MES, pH 6.0 and eluted with 1 M NaCl in 20 mM
MES, pH 6Ø Recombinant human NT-4 protein that stimulated E8 DRG outgr~wth was recovered in the 1 M NaCl wash.
The insoluble fraction was resuspended and homogenized in 83 ml 8 M guanidinium-HCl, 50 mM Tris-HCl, pH 8.5, 10 mM
NaCl, l~M EDTA. The insoluble fraction was dialyzed against 7 M urea, 50 mM Tris-Cl, pH 8.5, an~ loaded onto a Fast DEAE-Sepharose column equilibrated in 7 M urea, 50 mM
Tris-Cl, pH 8.5. The breakthrough fractions were collec~ed and dialyzed against 7 M urea, 100 mM histidine, pH 5.5, 1 -`
mM EDTA and loaded on a Fast S-Sepharose column eguilibrated in 7 M urea, 100 mM histidine, pH 5.5, 1 mM
EDTA, hNT-4 was eluted with a O-lM NaCl gradient in the r?
same buffer. Fractions containing hNT-4 were pooled and dialyzed against 100 mM histidine, pH ~.5, 1 mM EDTA.
Approximately 95% of the hNT-4 protein fractionated with the insoluble material.

9.3. PREPARATION OF ENRICHED MOTOR
NEU~ON CUL~URES
Embryos (E14) from Sprague-Dawley rats (HSD or Zi~ic-Miller) were used for these experiments. Pregnant rats SUBSTITUTE Si~EET

W092~2266~ PsCT/US92/05006 were sacrificed by carbon dioxide asphyxiation, and embryos were rapidly removed and placed in ice-cold medium for further dissection. Spinal cords were removed aseptically from rat embryos of 14 days gestation. The spinal cord was severed caudal to the bulb (at the level of the first dorsal root ganglion), freed of sensory ganglia and adhering menin~es. The cord was then subdsivided into ventral and mediodorsal segments for separate cultures.
The ventral spinal cord tissues were diced into small pieces and incubated in 0.1~ trypsin ~GIBC0) and 0.01%
deoxyribonuclease type 1 (SiSgma) in PBS at 37C for 20 minutes. Trypsin solution was then removed, rinsed and replaced with medium consisting of 45% Eagle's minimum essential medium (MEM), 45% Ham's nutrient mixture F12 (F12), 5% heat inactivated fetal calf serum (GIBCO) ~ 5%
heat inactivated horse serum (GIBC0), glutamine (2 mM), penicillin G (0.5 U/ml), and streptomycin (0.5 ug/ml). The tissue was then mechanically dissociated by gentle trituration t~rough a Pasteur pipet, and the supernatants were pooled and filtered through a nylon fiber (Nitex, Tetko; 40 ~m). The filtered cell suspension were then subjected to a modification of the fraction procedure described by Schnaar and Schaffner (1981, J. Neurosci.
~5:204-217). All steps were carried out at 4C.
Metrizamide was dissolved in F12:MEM (~:1) medium, and a discontinuous gradien~ was establi~hed which consisted of a 18~ metrizamide cushion (0.~ ml), 3ml of 17% metrizamide, 3ml of 12% metrizamide, and 3ml of 8% metrizamide was prepared. The filtered ventral spinal cord cell suspension (2.5ml) obtained as described above was layered over the step gradient, the tube was centrifuged at 2500g for 15 minutes using a swing-out rotor (Sorvall HB4).
~entrifugation resulted in three layers of cells: fraction I (at 0-8% interface), fraction II (at 8-12% interface), and fraction III tat 12-17% interface). The cells from SUBSTITUTE SHEET

: `
.

W092/22~ PCT/US92/0~006 _ 3 ~ 0 each interface were removed in a small volume (about lml), rinsed twice with serum-free defined medium consisting of 50% F12 and 50% MEM, supplemented with glutamine (2 mM), insulin (5 ug/ml), transferrin (lOOug/ml), progesterone (20 nM), putrescine (100 uM), and sodium selenite (30nM) (Bottenstein and Sato, 1979, PNAS 76:514-517). Viable cell `~-~
count was obtained by hemocytometer counting in the -~
presence of trypan blue. Fractionated ventral spinal cord cells (enriched with motorneurons) were then plated at a ;~
density of 100,000 cells/cm2 in 6 mm wells precoated with poly-L-ornithine (Sigma: 10 ~g/ml) and laminin (GIBCO:

10 ~g/ml). Treatment with NT-4 was given on the day of plating. Cultures were maintained in serum-free defined medium at 37C in 95% air/5% CO2 atmosphere at nearly 100% ~
relative humidity. On day 2 (48 hours), cells were ~-harvested for measurements of choline acetyltransferase (CAT; Fonnum, 1975, J. Neurochem. ~4:407-409.

9.4. RESULTS
The soluble fraction from the purification scheme of an induced overnight culture of pRG173/RFJ26 was assayed on E8 DRG explants. Figure 13 shows a dose-dependent stimulation of neurite outgrowth from the E8 DRG explants.
The ins~luble fraction was assayed on E8 dorsal root ganglia (D~G) explants and showed no bioactivity.
The recombinant human NT-4 protein recovered from the 1 m NaCl wash of the soluble fraction was assayed for activity in dissociated motor neuron cultures prepared as described sup~a at Example Section 9.3, and in other assays. Addition of recombinant human NT-4 at a 1:20 dilution resulted in a 3.6 fold increase in choline acetyltransfera~e activity in the motor neuron enriched culture 48 hours after treatment. The 3.6 fold increase was measured in relation to untreated (C-NT) and buffer (C-Buffer) controls (Figure 14). -SUBSTITUTE StlEET

.

-38,~ o PC~/US92l0~0~6 9.5. DISCUSSIQN
Subcloning the NT-4 coding region o~ bacteriophage HGi-2 downstream from the LAC W 5 and T7~1.1 promoters, the T7~1.1 ribosome binding site and the LamB signal sequence resulted in plasmid pRG173. Transformation o~ pRG173 into the ~. çoli strain RFJ26 and induction of large scale cultures of pRG17~/RFJ26 resulted in thQ expression of a biologically active form of recombinant human NT-4. The ability to express a biologically active form of human NT-4 in a recombinant prokaryotic expression system substantially increases the ease at which the production of human recombinant NT-4, peptides or derivatives thereof may be scaled up for both therapeutic and diagnostic appl~cations.
The present example thus teach~s that a DNA sequence encoding a human NT-4 is amenable to transcription and translation in a prokaryotic system such that human NT-4 is expressed and the biologi ally active form is amenable to purification schemes such that ths activity remains subsequent to purification~

.
10. DE~OSIT_OF MIÇROQ~GANISMS
The recombinant DNA molecules pRPN133 (hNGF), pRPN121 (hBDNFmyc) and pRPN14g (hBDNF) were deposited on June 7, 1991, pRG173 (NT-4) on October 28, 1991 and the recombinant bacte~iophag~ HG7-2 on August 22, 1991 with the American Type~Cultur~ Collection, 12301 Parklawn Drive, Rockville, Marylandi20852, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpcses of Pat~nt Procedure, and assigned the indicated accession number.

WO 92~22665 PCr/US92/0~006 3`~ Ll.lll~

RECOMBINANT SNA MOLECULE ATCC ACCESSION NO.
pRPN133 (hNGF) 75029 pRPN121 (hBDNFmyc~ 75028 pRPN149 (hBDNF) ~5027 pRG173 (hNT-4) 75131 HG7-2 Shuman NT-4 genomic clone) 75070 The present invention is not to be limited in scope by the deposited microorganisms or the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become appaxent to thoæe skilled in the art from the forega,ing description and accompany~ng figures. Such modifications are intended to fall within the scope of the appended claims. -~
Various publications have been cited herein which are incorporated by reference in thsir entireties.
While we have hereinbefore described a number of embodiments o f this invention, it is apparent that our basic embodiments can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appre~iated that the scope of this invention includes all alternative embodiments and variations which are defined in the f~regoing speeification and by the claims appended hereto;
and ~he in~ention is not to be limited by the specific embodiments which have been presented herein hy way of -:
example.

SUBSTITUTE SHEE~

Claims (89)

WE CLAIM:

1. A process for recovering a biologically active recombinant neurotrophin produced in a non-animal host cell culture comprising the step of solubilizing a recombinant neurotrophin in a solution comprising a strong denaturing agent, said solution being essentially free of reducing agents.

2. The process of claim 1 wherein the strong denaturing agent is a 4 M - 9 M guanidinium salt, a 4 M - 9 M alkali metal thiocyanate or 7 M - 9 M urea.

3. The process of claim 2 wherein the strong denaturing agent is 7 M - 9 M guanidinium HCl.

4. The process of claim 3 wherein the strong denaturing agent is 8 M guanidinium HCl.

5. The process of claim 1 further comprising the step of exchanging the strong denaturing agent for a weak denaturing agent.

6. The process of claim 5 wherein the weak denaturing agent is 4 M - 9 M urea.

7. The process of claim 6 wherein the strong denaturing agent is 7 M - 9 M guanidinium HCl and the weak denaturing agent is 6 M - 3 M urea.

8. The process of claim 7 wherein the strong denaturing agent is 8 M guanidinium HCl and the weak denaturing agent is 7 M urea.

9. The process of claim 1 wherein the solution further comprises a metalloproteinase inhibitor in an amount effective to inhibit degradation of the neurotrophin by proteases.

10. The process of claim 2 wherein the solution further comprises a metalloproteinase inhibitor in an amount effective to inhibit degradation of the neurotrophin by proteases.

11. The process of claim 10 wherein the solution further comprises 1 mM - 200 mM EDTA.

12. The process of claim 3 wherein the solution further comprises 5 mM - 80 mM EDTA.

13. The process of claim 4 wherein the solution further comprises 50 mM EDTA.

14. The process of claim 7 wherein the solution further comprises 5 mm - 80 mm EDTA.

15. The process of claim 8 wherein the solution further comprises 50 mm EDTA.

16. The process of claim 5 further comprising the steps of (1) adjusting the solution to comprise a basic amino acid or indole acetic acid at a concentration effective to maintain the solubility of the neurotrophin in a non-denaturing environment and (2) removing the weak denaturing agent.

17. The process of claim 6 further comprising the steps of (1) adjusting the solution to comprise a basic amino acid or indole acetic acid at a concentration effective to maintain the solubility of the neurotrophin in a non-denaturing environment and (2) removing the weak denaturing agent.

18. The process of claim 6 further comprising the steps of (1) adjusting the solution to comprise 20 mM -500 mM histidine and (2) removing the weak denaturing agent.

19. The process of claim 7 further comprising the steps of (1) adjusting the solution to comprise 20 mM -100 mM histidine and (2) removing the weak denaturing agent.

20. The process of claim 8 further comprising the steps of (1) adjusting the solution to comprise 20 mM -100 mM histidine and (2) removing the weak denaturing agent.

21. The process of claim 14 further comprising the steps of (1) adjusting the solution to comprise 20 mM -100 mM histidine and (2) removing the weak denaturing agent.

22. The process of claim 15 further comprising the steps of (1) adjusting the solution to comprise 20 mM -100 mM histidine and (2) removing the weak denaturing agent.

23. The process of claim 3, 4, 7, 8, 12, 13, 14, 15, 19, 20, 21 or 22 for recovering recombinant hNGF, hBDNF
or hNT-3.

24. The process of claim 23, for recovering recombinant hNGF having threonine substituted for serine as the second amino acid residue, full length hBDNF and full length hNT-3.

25. A process for producing a biologically active recombinant neurotrophin comprising the steps of:
culturing a non-animal host cell transformed with a recombinant DNA molecule comprising a controllable expression control sequence operatively linked to a DNA
sequence encoding a neurotrophin wherein said host cell is a heat shock regulatory gene mutant, under conditions such that said neurotrophin is produced by said host cell; and recovering the produced neurotrophin, wherein the recovered neurotrophin is biologically active.

26. The process of claim 25 wherein the host cell is an E. coli HtpR" lon mutant strain.

27. The process of claim 25 wherein the host cell is E. coli strain LC137.

28. The process of claim 26 wherein the expression control sequence comprises a .lambda. PL promoter.

29. The process of claim 28 for producing hNGF.

30. The process of claim 29 for producing an hNGF in which threonine replaces serine as the second amino acid residue.

31. The process of claim 25 comprising the step of culturing E. coli strain LC137 transformed with pRPN133 (hNGF)

32. The process of any of claims 26-28 for producing hNGF, hBDNF or hNT-3.

33. A process for producing a biologically active recombinant neurotrophin comprising the steps of:

culturing a non-animal host cell transformed with a recombinant DNA molecule comprising an expression control sequence operatively linked to a fused gene encoding, from 5' to 3', a signal sequence in-frame with a neurotrophin encoding sequence, under conditions such that said neurotrophin is produced by said host cell; and recovering the produced neurotrophin, wherein the recovered neurotrophin is biologically active.

34. The process of claim 33 wherein the host cell is E. coli and the signal sequence is LamB (SEQ ID
NO:1).

35. The process of claim 34 wherein the expression control sequence comprises a lacUV5 promoter and a T7 .PHI.1.1 ribosome binding site.

36. The process of claim 35 wherein the signal sequence is LamB1 (SEQ ID NO:2), LamB2 (SEQ ID NO:3), LamB3 (SEQ ID NO:4) or LamB4 (SEQ ID NO:5).

37. The process of claim 36 for producing full length hBDNF.

38. The process of claim 37 wherein the signal sequence is LamB1 (SEQ ID NO:2).

39. The process of claim 36 for producing hBDNFmyc.

40. The process of claim 39 wherein the signal sequence is LamB2 (SEQ ID NO:3) or LamB4 (SEQ ID NO:5).

41. The process of claim 33 comprising the step of culturing E. coli strain I?FW1330 transformed with pRPN149(hBNDF).

42. The process of any of claims 33-36 for producing hNGF, hBDNF or hNT-3.

43. Plasmid pRPN133(hNGF).

44. Plasmid pRPN121(hBDNFmyc).

45. Plasmid pRPN149(hBDNF).

46. A purified, homogeneous, biologically active neurotrophin recovered by the process of claim 1.

47. A purified, homogeneous, biologically active neurotrophin recovered by the process of claim 5.

48. A purified, homogeneous, biologically active neurotrophin recovered by the process of claim 9.

49. A purified, homogeneous, biologically active neurotrophin recovered by the process of claim 10.

50. A purified, homogeneous, biologically active neurotrophin recovered by the process of claim 16.

51. A purified, homogeneous, biologically active neurotrophin recovered by the process of claim 17.

52. The purified, homogeneous, biologically active neurotrophin of any of claims 46-51 which is hNGF, hBDNF or hNT-3.

53. The purified, homogeneous, biologically active neurotrophin of claim 52 which is an hNGF in which threonine replaces serine as the second amino acid residue.

54. The purified, homogeneous, biologically active neurotrophin of claim 52 which is a full length neurotrophin.

55. A modified LamB signal sequence gene comprising a DNA molecule encoding a modified LamB signal sequence selected from the group consisting of LamB1 (SEQ
ID NO:2), LamB2 (SEQ ID NO:3), LamB3 (SEQ ID NO:4) and LamB4 (SEQ ID NO:5).

56. A modified LamB signal sequence gene comprising a DNA molecule encoding a LamB signal sequence containing fourteen hydrophobic amino acids in the hydrophobic core region of the LamB signal sequence.

57. The gene of claim 56 wherein the hydrophobic core region of the LamB signal sequence contains fourteen hydrophobic amino acids, comprising a peptide having the sequence LAVL (SEQ ID NO:6).

58. The process of claim 3 for recovering biologically active recombinant neurotrophin-4.

59. The process of claim 58 for recovering biologically active human neurotrophin-4.

60. The process of claim 59 for recovering biologically active human neurotrophin-4 encoded by a DNA
sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070, and illustrated in Figure 11 (SEQ ID NO:22).

61. The process of claim 59 for recovering biologically active human neurotrophin-4 encoded by a DNA
sequence as contained in DNA plasmid pRG173 as deposited with the ATCC and assigned accession number 75131.

62. The process of claim 25 wherein said recombinant DNA molecule encodes neurotrophin-4.

63. The process of claim 25 wherein said recombinant DNA molecule encodes human neurotrophin-4.

64. The process of claim 63 wherein the human neurotrophin-4 is encoded by a DNA molecule having a sequence as contained in bacteriophage HG7-2, as deposited with the ATCC and assigned accession number 75070 and illustrated in Figure 11 (SEQ ID NO:22).

65. The process of claim 63 wherein the human neurotrophin-4 is encoded by a DNA molecule having a sequence as contained in E. coli plasmid vector, pRG173, as deposited with the ATCC and assigned accession number 75131.

66. The process of claim 25 comprising the step of culturing E. coli strain RFJ26 transformed with plasmid vector, pRG173, as deposited with the ATCC
and assigned accession number 75131.

67. The process of claim 33 wherein said recombinant DNA molecule encodes neurotrophin-4.

68. The process of claim 67 wherein said recombinant DNA molecule encodes human neurotrophin-4.

69. The process of claim 68 wherein the human neurotrophin-4 is encoded by a DNA molecule having a sequence as contained in bacteriophage HG7-2, as deposited with the ATCC and assigned accession number 75070 and illustrated in Figure 11 (SEQ ID No: 22).

70. The process of claim 68 wherein the human neurotrophin-4 is encoded by a DNA sequence as contained in E. coli plasmid vector, pRG173, as deposited with the ATCC
and assigned accession number 75131.

71. The process of claim 33 comprising the step of culturing E. coli strain RFJ26 transformed with coli plasmid vector, pRG173, as deposited with the ATCC
and assigned accession number 75131.

72. The DNA plasmid vector pRG91.

73. The DNA plasmid pRG173 as deposited with the ATCC and assigned accession number 75131.

74. A purified, homogeneous, biologically active neurotrophin-4 recovered by the process of claim 1.

75. The purified homogenous, biologically active neurotrophin-4 of claim 74 which is human neurotrophin-4.

76. The purified, homogeneous, biologically active neurotrophin-4 of claim 75 encoded by a DNA sequence as contained in the bacteriophage HG7--2, as deposited with the ATCC and assigned accession number 75070, and illustrated in Figure 11 (SEQ ID NO:22).

77. The purified, homogeneous, biologically active neurotrophin-4 of claim 75 encoded by a DNA sequence as contained in E. coli plasmid vector, pRG173, as deposited with the ATCC and assigned accession number 75131, as illustrated in Figure 12.

78. A process for producing biologically active human NT-4 comprising growing a bacterium containing a recombinant nucleic acid encoding human NT-4 under conditions such that the human NT-4 is produced by the bacterium, and recovering the produced human NT-4, wherein the recovered human NT-4 is biologically active.

79. The process of claim 25 comprising the step of culturing E. coli strain RFJ26 transformed with E. coli plasmid vector, pRG173, as deposited with the ATCC and assigned accession number 75131.

80. The process of claim 68 wherein the human neurotrophin-4 is encoded by a DNA molecule having a sequence as contained in E. coli plasmid vector, pRG173, as deposited with the ATCC and assigned accession number 75131.

81. The purified, homogeneous, neurotrophin-4 of claim 75 encoded by a DNA molecule having a sequence as contained in E. coli plasmid vector, pRG173, as deposited with the ATCC and assigned accession number 75131.

82. The process of claim 34 wherein the signal sequence is LamB1 (SEQ ID NO:2), LamB2 (SEQ ID NO:3), LamB3 (SEQ ID NO:4) or LamB4 (SEQ ID NO:5).

83. A fusion gene for expression of a neurotrophin protein comprising from 5' to 3', an expression control DNA sequence operatively linked to the signal sequence of claim 55, that is linked in-frame with a DNA sequence encoding a neurotrophin protein.

84. A fusion gene for expression of a neurotrophin protein comprising from 5' to 3', an expression control DNA sequence operatively linked to the signal sequence of claim 56 that is linked in-frame with a DNA sequence encoding a neurotrophin protein.

85. A fusion gene for expression of a neurotrophin protein comprising from 5' to 3', an expression control DNA sequence operatively linked to the signal sequence of claim 57, that is linked in-frame with a DNA sequence encoding a neurotrophin protein.

86. A modified LamB signal sequence gene comprising a DNA sequence encoding a modified LamB signal sequence having a hydrophobic core region of 11-16 amino acid residues.

87. The process of claim 36 wherein the signal sequence is modified to contain fourteen hydrophobic amino acids in the hydrophobic core.

88. The gene of claim 86 which is LamB1 (SEQ ID
NO:2) or LamB2 (SEQ ID NO:3).

89. The gene of claim 86 which is LamB3 (SEQ ID
NO:4) or LamB4 (SEQ ID NO:5).