GB2071671A

GB2071671A - Polypeptide production

Info

Publication number: GB2071671A
Application number: GB8108136A
Authority: GB
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 1980-03-17
Filing date: 1981-03-16
Publication date: 1981-09-23
Also published as: AT378004B; ATA122281A; DE3110031A1; DE3153267A1; GB2071671B; BE887991A; ES8306177A1; ES8205432A1; JPS56167698A; ES510007A0; IT8167359A0; FR2478125A1; NL8101289A; FR2478125B1; IT1144705B; ES500397A0; SE8101665L; CA1198385A

Abstract

Compositions and methods featuring, in one aspect, a method of providing a fused gene having a promoter located an optimal distance from the translation start site, the method including providing a fused gene having a region of a gene coding for a eukaryotic or prokaryotic polypeptide fused to a region of a gene coding for an assayable polypeptide, inserting a portable promotor at varying distances in front of the translation start site, cloning the fused genes into microorganisms, selecting those producing the greatest amount of assayable polypeptide, and reconstituting the gene for the eukaryotic or prokaryotic protein.

Description

SPECIFICATION Polypeptide production This invention relates to providing gene fusions producing an optimal amount of polypeptide.

In a publication by Backman and Ptashne, Cell, Vol. 13, pages 65-71(1978), there was described a method for producing unfused protein by creating, in front of the gene for the protein, a hybrid ribosome binding site consisting of a Shine Dalgarno sequence (attached to a portable promoter), at least two other base pairs, and the ATG (translation start site) of the gene for the protein, and then cloning the fused gene into a microorganism. The quantity of protein produced is in part a function of the length of the hybrid ribosome binding site; that is, the distance, in terms of base pairs, between the Shine-Dalgarno sequence and the translation start site. This distance is varied by varying the position of the portable promoter, as described in our earlier application, supra.

When the hybrid ribosome binding site is of some optimal length, protein production is maximized. One method of determining the promoter position which yields this optimal ribosome binding site is to use a standard RIA technique to measure protein production.

However, standard techniques are cumbersome for some polypeptides, and unavailable for others.

The limitation of standard protein assaying methods can also impede the determination of the efficacy of a naturally occurring ribosome binding site. Similarly, these limitations can make it difficult or impossible to detect a mutation, spontaneous or induced, which results in increased polypeptide production.

We have discovered a new method for determining the amount of eukaryotic or prokaryotic polypeptide produced by a microorganism containing a desired gene coding for the polypeptide ("polypeptide", as used herein, includes proteins and small polypeptides). The method involves fusing an amino terminal region of a sample of the desired gene, which region codes for a fragment of the eukaryotic or prokaryotic polypeptide, to a carboxy terminal gene region coding for a different, assayable, polypeptide fragment. The fused gene is cloned into a microorganism, where it encodes an assayable hybrid polypeptide product having an amino-terminal fragment of the desired polypeptide, and a carboxy-terminal fragment of the assayable polypeptide. The amount of assayable hybrid polypeptide is measured; this amount is directly proportional to the amount of eukaryotic or prokaryotic polypeptide.

The above principles and techniques are also used in another aspect of the invention, a method of providing, for a gene coding for a desired eukaryotic or prokaryotic polypeptide, a sample of the gene having one or more mutations causing the mutated gene to code for a greater amount of the desired polypeptide than non-mutated samples of the gene. The method involves first providing samples of a fused gene including a carboxy-terminal gene region coding for an assayable polypeptide fragment fused to an amino-terminal gene region coding for a fragment of the desired polypeptide. The fused genes are cloned into microorganisms, where they produce assayable hybrid polypeptide.Any fused gene having one or more spontaneous mutations causing it to produce more of the hybrid polypeptide than non-mutated samples is selected, and then, from this fused gene, the gene for the desired polypeptide is reconstituted by any of the usual in vitro or in vivo methods. The gene is at this point ready to be cloned into a microorganism, where it can direct the production of higher levels of the desired polypeptide in pure form.

The above method can be modified by mutagenizing the microorganisms after the initial cloning step. This can be done using standard mutagens, e.g., X-rays or chemical mutagens such as nitrosoamines.

The mutation selecting method is particularly useful with regard to fusions which fail to produce iarge amounts of polypeptide no matter where the promoter is located. Successive mutations can eventually yield samples capable of efficient polypeptide production.

The above principles and techniques are also used in another aspect of the invention, a method for providing genes having maximally efficient hybrid ribosome binding sites. The method involves providing, as described above, samples of a fused gene including a gene region coding for a fragment of the desired eukaryotic or prokaryotic polypeptide fused to a gene region coding for a different, assayable, polypeptide fragment. The fused gene encodes an assayable hybrid polypeptide product having an amino-terminal fragment of the desired polypeptide, and a carboxy-terminal fragment of the assayable polypeptide. A portable promoter is placed at varying distances in front of this fused gene, and the fusions having the promoter in the position producing the most assayable hybrid polypeptide product are selected.The gene for the desired polypeptide is then reconstituted, and can produce high levels of unfused polypeptide.

In the drawings, Fig. 1 shows a schematic diagram of a generalized fused gene of the invention, Figs. 2 and 3 show diagrammatic representations of the construction of two fused genes of the invention, Fig. 4 shows a diagrammatic representation of in vitro gene reconstitution, and Fig. 5 shows a diagrammatic representation of in vivo gene reconstitution.

Any carboxy terminal gene region coding for an assayable polypeptide fragment can be used in accordance with any aspect of the invention, and the gene region coding for the desired polypeptide fragment can be inserted in the gene region for the different assayable polypeptide fragment in any position which does not destroy assayability.

To eliminate concern with whether or not the inserted desired polypeptide fragment will destroy assayability, prior to that insertion, a gene or gene region can be used to the gene region for the assayable polypeptide fragment to serve as the insertion site.

Referring to Fig. 1, a fused gene of the invention which facilitates gene insertion as described above includes from right to left, a first carboxy-terminal gene region coding for an assayable polypeptide fragment fused to a second amino-terminal gene region bearing a restriction site. (Herein, "left" and "right" are used to indicate positions which are transcribed, respectively, earlier and later). If that region does not naturally bear such a site, a DNA linker sensitive to one or more restriction enzymes is inserted in the region.

The natural restriction site or DNA linker serves as the site for the insertion into the second gene region of a third gene region, an amino-terminal region of a gene for a fragment of any desired prokaryotic or eukaryotic polypeptide. This region may have its own translation start site (ATG), or it may be lacking such a site. In the latter instance, such a site can be fused to the left end of the desired gene region.

In front of the third gene region is a promoter which includes a transcription start site and a Shine-Dalgarno sequence. This promoter is either the native promoter or, in the method involving varying the promoter position, a portable promoter.

There are always at least two base pairs between the Shine-Dalgarno sequence and the translation start site. In the case of the assay method not employing a portable promoter, they are of course part of the native DNA strand which includes the desired gene. In the method involving varying promoter position, these base pairs are either originally attached to the promoter's Shine Dalgarno sequence, or to be desired gene, or some can be attached to each.

If the base sequence of the gene region for the desired polypeptide fragment is known to the extent that the correct reading frame for that gene region is known, a DNA linker, if one is necessary, is positioned such that the desired gene region, after insertion, will be in phase with the first gene region. If the sequence is not adequately known, the desired gene region can be inserted into three different fusions, having differently positioned linkers. The linkers can be positioned anywhere on the second gene region, but only three positions are needed to cover all three possible reading frames. The linker position which allows translation of the particular gene region desired to proceed in frame into the first gene region is the one to be used with that gene region.

To provide the fused genes having the optimally-placed promoter, a portable promoter is attached to samples of the fusions described above at varying distances from the location of the translation start site. The samples of the fused genes, attached to promoters, are cloned into microorganisms. The fused genes bearing the promoter fragment at the optimal distance from the translation start site are recognized and selected on the basis of their production of the greatest amount of assayable hybrid polypeptide.

The gene for the desired eukaryotic or prokaryotic polypeptide, with the optimally placed promoter, is then reconstituted. Reconstitution can be carried out on the same fusion sample which was used for the polypeptide assay, or it can be performed on a sample of that fusion set aside before the polypeptide assay was carried out.

Also, because the optimal promoter distance is known after the polypeptide assay has been done, a portable promoter can be inserted the optimal distance from another sample of the fusion, and reconstitution can then be performed on that fusion. After reconstitution, the gene is ready to direct the production, in a microorganism, of a maximum amount of the desired polypeptide in pure form.

The following specific example is intended to illustrate more fully the nature of the present invention without acting as a limitation upon its scope.

EXAMPLE Three plasmids, pLG 200, pLG 300, and pLG 400 were constructed. Each has a site, vulnerable to a restriction enzyme, in a different translational reading frame on a region of the lacl gene. Each of the three plasmids also contains a region of the IacZ gene which codes for an assayable fragment of the polypeptide ,B- galactosidase. Plasmids pLG 300 and pLG 400 were constructed from pLG 200, which was in turn constructed in the following manner, as shown in Fig. 2.

First, plasmid pLG 2, containing an intact lacllacP-lacZ gene fusion, was constructed as follows An Eco RI fragment from pMC4 containing the intact lacl gene, the lac promoter, and the amino terminal region of lacZ was ligated to an Eco RI blunt ended fragment (from A112, described in Ptashne in The Operon, Miller et al., eds. (Cold Spring Harbor Laboratory, New York 1978)) containing the carboxy terminal region of JacZ to create a fragment with an intact lacl-lacP-lacZ region. This fragment was inserted into a backbone containing pBR 322 DNA from the RI site to the Pvull site, yielding plasmid pLG 2.

Next, a lacl-lacZfused gene was crossed onto pLG 2 by in vivo recombination to yield plasmid pLG 5. This plasmid was then partially digested with Pvull, and the amino terminal region of the lacl region was replaced with a Hin d III linker to yield pLG 200 which, when opened at the Hin d Ill site, provides for one of the three possible translational reading frames.

Plasmid pLG 300, capable of providing a second reading frame, was constructed from pGL 200 as follows. pLG 200 was cut with Hin d III, the ends were filled in the DNA polymerase, and a Bam linker inserted, yielding pLG 300.

To provide the third reading frame, pLG 300 was cut with Bam, filled in, and Hin d Ill linker was inserted to yield pLG 400.

Referring to Fig. 3, a region of a gene for a desired polypeptide, rabbit /3-globin, was inserted into plasmid pLG 300 by ligating a filled-in Pst Bam globin fragment from pGL 6 to a filled-in Pst Bam backbone fragment of pLG 300 to yield pLG 302.

A lac promoter fragment was inserted varying distances in front of the translation start site of the fused gene, according to the method described in Ptashne et al. Serial No. 3,102, supra. pLG 302 was opened with Hin d Ill, resected with exonuclease Ill and S1 (or Bal 131), and then cut with Pst so that a promoter fragment with a Pst end and a blunt end could be inserted.

Plasmids with promoters at varying distances from the start of the fused gene were cloned into E. coli and grown on indicator agar which changes color to a degree depending on the level of p-galactosidase produced by the bacteria. Those colonies producing the greatest color change were selected as the ones having the promoter optimally positioned.

Referring to Fig. 4, the intact !;-globin gene was reconstituted in vitro as follows. From the selected plasmids, a Rl-Sau 3A fragment containing the lac promoter and the amino terminal coding portion of the ,-globin gene was prepared. This fragment was ligated to a Bam-Alu fragment from p/3G1 (Efstratiadis et al. (1977) Cell, 10, 571-585) containing the carboxy terminalcoding region of the 83-globin gene. The reconstituted gene was then inserted into an Ri--Pvulli backbone of pBR 322. This reconstituted gene, when cloned into E. coli, which was then cultured, directed the production of maximal quantities of the desired rabbit ,B-globin.

Referring to Fig. 5, in another sample, the ss-globin gene could be reconstituted in vivo as follows. The fusion bearing the optimally-placed promoter was inserted into a plasmid having a marker for ampicillin resistance. This plasmid was then crossed with another plasmid carrying the missing portion of the gene for /3-globin, plus a small region of overlap to provide homology for genetic recombination. The latter plasmid bore no lac promoter, but it did carry a marker for tetracycline resistance. The desired recombination separated the lac promoter from the i3- galactosidase gene; this recombination was thus identifiable among other recombinants exhibiting both drug resistances by its lac-phenotype.

The reconstitution method described herein are the same ones to be used after employing the method of the invention to select desirable mutants which have arisen spontaneously or which have been induced by means of a mutagen.

Other embodiments are within the following claims. For example, the gene region for an assayable polypeptide can be fused to genes other than lacl; an example is the t antigen of Simian virus, discussed in Robert et al. (1979), Proc. Natl.

Acad. Sci., U.S.A., 76, 5596-5600. Other suitable genes, e.g. Male and lamB are described in Bassford et al., in The Operon, Miller et al., eds., pp. 245-261 (Cold Spring Harbor Laboratory, New York 1978). Also, among the genes for polypeptides having assayability in the carboxyterminal region which can be used in place of the 5-galactosidase gene are, for example, the E. coli trpC and trpD genes, which code for two polypeptides, each having two specific enzymatic activities in tryptophane biosynthesis; and yeast genes which code for enzymatically-active polypeptides. For example, one such yeast gene codes for a polypeptide having three enzymatic activities involved in histidine biosynthesis.

Claims

1. A fused gene comprising: a first, carboxy terminal gene region coding for an assayable polypeptide fragment, a second, amino terminal gene region fused to said carboxy terminal gene region, said amino terminal gene region having a restriction site, a third, amino terminal gene region coding for a fragment of a eukaryotic or prokaryotic polypeptide, said third gene region being located at said restriction site and, in front of said third gene region, a translation start site and a portable promoter having a transcription state site.

2. The fused gene of claim 1, wherein said first gene region is a region of the gene coding for co/i cold j3-galactosidase, and said second gene region is a region of the gene coding for E. coli repressor.

3. A method of determining the amount of a eukaryotic or prokaryotic polypeptide produced by a microorganism containing a desired gene coding for said polypeptide, which method comprises fusing an amino terminal region of a sample of said desired gene to a carboxy terminal gene region coding for a different, assayable polypeptide fragment thereby to produce a fused gene coding for an assayable hybrid polypeptide, cloning said fused gene into a microorganism, and measuring the amount of said assayable hybrid polypeptide produced by said fused gene in said microorganism, said amount being directly proportional to the amount of said eukaryotic or prokaryotic protein.

4. A method of providing, for a fused gene coding for a desired eukaryotic or prokaryotic polypeptide, a sample of said fused gene having its portable promoter located an optimal distance from its translation start site comprising the steps of providing samples of a fused gene comprising a carboxy terminal gene region coding for an assayable polypeptide fragment fused to an amino terminal gene region coding for a fragment of said desired eukaryotic or prokaryotic polypeptide, said fused gene being capable of coding for an assayable hybrid polypeptide, inserting said portable promoter at varying distances in front of the translation state site of said samples of said fused gene, cloning said samples of said fused gene into microorganisms, assaying said assayable hybrid polypeptide produced by said fused genes in said microorganisms, selecting said fused gene producing the greatest amount of said assayabie hybrid polypeptide, and reconstituting, from said selected fused gene, the gene coding for said desired eukaryotic or prokaryotic polypeptide.

5. A method of providing, for a gene coding for a desired eukaryotic or prokaryotic polypeptide, a sample of said gene having one or more mutations causing said mutated gene to code for a greater amount of said desired polypeptide compared to non-mutated samples of said gene, comprising the steps of providing samples of a fused gene comprising a carboxy terminal gene region coding for an assayable polypeptide fragment fused to an amino terminal gene region coding for a fragment of said desired eukaryotic or prokaryotic polypeptide, said fused gene being capable of coding for an assayable hybrid polypeptide, cloning said samples of said fused gene into microorganisms, assaying said assayable hybrid polypeptide produced by said fused genes in said microorganisms, selecting the mutated fused gene producing a greater amount, compared to non-mutated samples of said fused gene, of said assayable hybrid polypeptide, and reconstituting, from said selected fused gene, the gene coding for said desired eukaryotic or prokaryotic polypeptide.

6. The method of claim 5, further comprising, following said step of cloning said fused gene into said microorganisms, the step of mutagenizing said microorganisms.

7. The method of claim 4, wherein said gene region coding for said assayable polypeptide fragment is a region of the E. coli gene coding for /3-galactosidase.

8. The method of claim 5 or 6, wherein said gene region coding for said assayable polypeptide fragment is a region of the E. colt gene coding for ,8-galactosidase.

9. A fused gene substantially as hereinbefore described in the Example.

10. A method of determining the amount of eukaryotic or prokaryotic polypeptide produced by a microorganisms substantially as hereinbefore described in the Example.

11. A method of preparing a fused gene substantially as hereinbefore described in the Example.

1 2. A method of preparing a gene housing one or more mutations substantially as hereinbefore described in the Example.

13. Polypeptide or protein produced by an organism housing a fused gene as claimed in claim 1.