IE911322A1 - Process for producing foreign proteins in streptomyces - Google Patents

Abstract

Gene structures which code for the signal sequence and approximately the first ten amino acids of tendamistat as well as a required protein are expressed in high yield in streptomyces host cells, and the fusion proteins are secreted into the medium.

Description

HOECHST AKTIENGESELLSCHAFT HOE 90/F 122 Dr.KL/gm Description Process for producing foreign proteins in streptomyces The European Patent Application with the publication number (EP-A) 0,289,936 discloses the production of fusion proteins by coupling the structural gene for the desired protein to the 3'-end of the coding strand of the optionally modified tendamistat gene, expressing this genetic structure in a streptomyces host cell and isolating the secreted fusion protein from the supernatant. In a preferred embodiment the tendamistat gene is truncated at the 3'-end. For the truncation, the cleavage sites for the restriction enzyme BstEU in the region of triplets 31 and 32, Stul in the region of triplets 43 and 44, and Sau3A in the region of triplets 52 and 53 are used.

In a further development of this inventive concept it has already been proposed to produce a fusion protein in which a truncated proinsulin whose C-chain is composed of only one or two lysine residues (mini-proinsulin) follows the tendamistat portion. Another further develop20 ment which has been proposed is to truncate the tendamistat portion, too, in fusion proteins of this type (EP-A 0,367,163 published on May 9,1990).

Surprisingly, it has now been found that fusion proteins with a very short tendamistat portion are stable in streptomyces cells and are secreted into the medium. The fusion proteins obtained in this way behave like mature proteins because of the very short tendamistat chain and generally are present in the medium in the correct tertiary structure.

EP-A 0,177,827 discloses a synthetic signal sequence for transporting proteins in expression systems, wherein the DNA is virtually identical to a natural signal sequence but has one or more cleavage sites for endonucleases, - 2 which are not contained in the natural DNA. When the gene for the protein to be transported is coupled to such a DNA sequence, this fusion gene is incorporated into a vector, and a host cell which transports the expressed protein out of the cytoplasm is transformed therewith, it is possible to produce eukaryotic, prokaryotic or viral proteins in prokaryotic and eukaryotic cells. Using the periplasmic protein alkaline phosphatase as an example, it is shown that it is advantageous in the expression in E. coli to place the codons for about the first 40 amino acids of alkaline phosphatase immediately downstream of the pre-sequence and upstream of the structural gene for the desired protein. However, in many cases even fewer additional amino acids are sufficient, for example about , preferably about 5. About 90% of a corresponding fusion protein with simian proinsulin was transported into the periplasmic space.

It has also been proposed (WO 91/03550, published March 21, 1991) to produce fusion proteins by constructing a mixed oligonucleotide which codes for the ballast portion of the fusion protein, introducing this oligonucleotide into a vector in such a way that it is functionally coupled to a regulatory region and the structural gene for the desired protein, transforming suitable host cells with this plasmid population obtained in this way and selecting those clones which show a high yield of coded fusion protein. The oligonucleotide preferably consists of 4 to 12, in particular 4 to 8, triplets in this case.

It has already been attempted to produce fusion proteins with a short ballast portion. Thus, a gene fusion which codes for a fusion protein from the first 10 amino acids of 0-galactosidase and somatostatin has, for example, been produced. However, it became apparent that this short ^-galactosidase fragment was not sufficient to protect the fusion protein from digestion by endogenous host proteases (US-A 4,366,246, column 15, paragraph 2). - 3 Accordingly, fusion proteins whose ballast portion consists of a 0-galactosidase fragment having more than 250 amino acids are described in EP-A 0,290,005 and 0,292,763.

However, fusion proteins of about the first 10 aminoterminal amino acids of tendamistat and a desired protein, for example a proinsulin, are, in fact, unexpectedly stable in streptomyces host cells and are secreted into the medium, from which they can be isolated in high yields. Surprisingly this is also true for relatively small proteins such as mini-proinsulins.

About 10 amino acids is intended to mean in this case that even fewer amino acids are suitable, for example the first 7 N-terminal amino acids of tendamistat, but preferably not more than 10. Fusion proteins in whose tendamistat portion proline is present in position 7 and/or 9 (as in the natural sequence) are preferred. However, it is, of course, possible to choose a larger tendamistat ballast portion in accordance with the embodiments already known or proposed, the advantage of low ballast being lost more and more, of course.

It is possible and even advantageous to vary the natural amino acid sequence of the tendamistat portion, i.e. to exchange or delete amino acids, or to insert amino acids which do not occur in the natural amino acid sequence.

Furthermore, it is possible to vary the amino acid sequence in the signal peptide.

Particularly advantageous fused constructions can be readily determined by simple preliminary experiments if the concept of the invention is known.

It is furthermore possible to realise the concept of the invention also in other Gram-positive bacterial cells, for example in bacillus or staphylococcus cells using signal sequences which are recognized by these hosts. - 4 The fusion proteins obtained according to the invention are present in the medium in a dissolved form, which has many advantages in processing and purification. Thus, enzymatic processing with cleaving of the ballast portion can, for example, take place directly on the secretion product, and working-up steps, such as the ones necessary for insoluble fusion proteins, do not have to be carried out. It is also possible to carry out concentration or purification processes, for example affinity chroma10 tography but also ultrafiltration, precipitation, ion exchange chromatography, adsorption chromatography, gel filtration or high-pressure liquid chromatography, first, before further processing.

In the examples which follow the invention is illustrated in more detail.

The starting material for the plasmid constructions is plasmid pKK500 which was proposed in EP-A 0,367,163. This plasmid differs from plasmid pKK400 known from EP-A 0,289,936 in that the proinsulin gene is replaced by an analogous gene which, instead of the C chain, merely encodes the amino acid lysine, and in that a terminator sequence is inserted immediately downstream of this mini-proinsulin gene. Tables 1 and 2 from EP-A 0,367,163, in which the mini-proinsulin gene and the terminator sequence, respectively, are shown, are enclosed as an annex to the description.

The plasmids pKK400 and pKK500 contain a Xmalll cleavage site in the signal sequence of the α-amylase inhibitor gene (in the region of triplets -5 to -7).

Example 1 Plasmid pKK500 is opened up with the restriction enzymes EcoRI and Xmalll, and the large fragment is separated by gel electrophoresis on a 0.8% agarose gel and isolated by electroelution. This fragment is ligated with the DNA - 5 fragment (1) (SEQ ID NO:1) -5 -1 Ala Gly Pro Ala Ser Ala GCC GGG CCG GCC TCC GCC CCC GGC CGG AGG CGG (Xmalll) Asp Thr Thr Val Ser Glu Pro GAC ACG ACC GTC TCC GAG CCG 3' CTG TGC TGG CAG AGG CTC GGC TTA A 5' (EcoRI) which has been synthesized by the phosphoramidite method, 5 and the ligation mixture is transformed into E. coli.

Plasmid pKK510 is obtained. This plasmid encodes a preproinsulin in which the signal sequence of tendamistat is followed by the first 7 amino acids of tendamistat which are followed by the mini-proinsulin chain.

Example 2 In analogy with the process described in EP-A 0,289,936 for transferring plasmid pKK400 into expression plasmid pGFl, plasmid pKK510 is transferred into expression plasmid pKFl: The isolated plasmid DNA of pKK510 is cut with the restriction enzymes SphI and Sstl, and the small fragment with the fusion gene is isolated. The commercial expression plasmid piJ 702 (obtainable from John Innes Foundation, Norwich, England) is cut with the same enzymes and the large fragment is isolated. These two isolated fragments are ligated, the ligation mixture transformed into S. lividans TK24 and the plasmid is isolated from the thiostrepton-resistant white (i.e. not capable of forming melanin) transformants. Clones which carry the introduced insert are tested for the formation of fusion proteins in a shake culture. - 6 The encoded fusion protein is expressed in a manner known per se: if the transformed strain is incubated in a shaken flask at above 25 "C for 4 days and the mycelium is separated from the culture solution by centrifugation, it is possible, after electrophoresis of 20 μΐ of culture filtrate in a 15% polyacrylamide gel, to visualize by dyeing with *COOMASSIE Blue the fusion protein formed in the culture supernatant as an additional protein band which does not occur in a control experiment in which the strain was transformed only with piJ 702.

If the culture filtrate is treated with lysyl endoproteinase, it is possible to detect de-(B30)-Thrinsulin, which is verified by an authentic control on gel electrophoresis .

Furthermore, it is possible to detect the fusion protein in the culture filtrate with insulin antibodies either in an immunoblot or with an insulin RIA.

Example 3 The procedure according to Examples 1 and 2 is carried out, but the synthetic fragment (2) with the SEQ ID NO:2 -5 -1 Ala Gly Pro Ala Ser Ala 5' G GCC GGG CCG GCC TCC CCC 3 ' CCC GGC CGG AGG CGG (Xmalll) Asp Thr Thr Val Ser Glu Pro Asp Pro GAC ACG ACC GTC TCC GAG CCC GAC CCG 3’ CTG TGC TGG CAG AGG CTC GGG CTG GGC TTA A 5' (EcoRI) is used and the plasmids pKK320 and pKF2, respectively, are obtained in this way. - 7 These plasmids encode a fusion protein which differs from the one according to Examples 1 and 2 in that the first 7 amino acids of tendamistat are followed by asparagine (instead of the natural amino acid alanine) and that this is followed by the ninth amino acid in tendamistat, proline. By exchanging alanine for asparagine, an additional positive charge is therefore introduced into the ballast portion of the fusion protein. Surprisingly, yields about 20 to 30% higher than in Example 2 are obtained.

Example 4 If the procedure according to Examples 1 and 2 is carried out, but the synthetic fragment (3) with the SEQ ID NO:3 -5 -l Ala Gly Pro Ala Ser Ala ’ G GCC GGG CCG GCC TCC GCC 3' CCC GGC CGG AGG CGG (Xmalll) Asp Thr Thr Val Ser Glu Pro Ala Pro GAC ACG ACC GTC TCC GAG CCC GCA CCG 3' CTG TGC TGG CAG AGG CTC GGG CGT GGC TTA A 5’ is used, the plasmids pKK330 and pKF3, respectively, are obtained. These plasmids differ from those according to Examples 1 and 2 in that they encode the first 9 natural amino acids of tendamistat. In comparison with Example 2, yields about 10% higher are obtained.

Example 5 The fusion protein encoded by pKK500 contains between the tendamistat portion and the B chain of proinsulin a linker sequence which codes for the amino acids - β Asn-Ser-Asn-Gly-Lys. This terminal Lys and the Lys representing the C chain are replaced by Arg as described below. In this procedure, the single Styl cleavage site in the region of codons B30 to Al in the proinsulin sequence is used.

Isolated plasmid DNA from pKK500 is cut using Styl, digested with SI nuclease to remove protruding ends and the excess nuclease is extracted using phenol-chloroform. The linearized plasmid is then subsequently cut with EcoRl, and the large fragment is electrophoretically separated off and isolated by electroelution. This fragment is ligated with the synthetic fragment (4) (SEQ ID NO:4) BI 10 Asn Ser Asn Gly Arg Phe Val Asn Gin His Leu Cys Gly Ser His AAT TCG AAC GGC CGC TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC GC TTG CCG GCG AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG (EcoRl) 30 Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG B30 C(B31) Tyr Thr Pro Lys Thr Arg TAC ACC CCC AAG ACC CGC ATG TGG GGG TTC TGG GCG and the ligation mixture is transformed into E. coli. The desired clones are tested by restriction analysis of the plasmid contained, using the newly developed SetII cleavage site. Furthermore, the entire Sphl-Sstl fragment is sequenced.

In order to express the encoded fusion protein, the - 9 fragment, which has been checked by sequence analysis, is ligated into the vector piJ 702, which has been cut with the same enzymes, resulting in the expression vector pGF4.

The secreted fusion protein encoded by pGF4 can be detected, on the one hand, by the a-amylase inhibitor plate test (EP A 0,161,629, Example 3) and, on the other hand, from the supernatant of the shake culture in analogy with Example 2.

Example 6 If fragment (4) is, in analogy with Example 5, inserted into the vectors pKK510, 520 and 530, the vectors pKK610, 620 and 630 are obtained. The incorporation of the respective Sphl-Sstl fragments with the coding sequence for the fusion proteins into the vector piJ 702 results in the expression vectors pKFll, 12 and 13. The expression of the secreted fusion proteins is tested in analogy with Example 2.

Example 7 In order to increase the expression of derivatives of the plasmid piJ 702, the melanin promoter is deleted therefrom by digestion with Pstl and SphI and is replaced by the synthetic fragment (5) (SEQ ID NO:5) Pstl Bell 30 40 50 CTGCAGTGATCAGGGGGACCCTTGTCCGAATTTCCGTTACGGGTTTGGGTGGTAGGG GACGTCACTAGTCCCCCTGGGAACACGCTTAAAGGCAATGCCCAAACCCACCATCCC SphI 70 80 25 ACGCACCCGAAGAGGAGGCCCCAGCATGC TGCGTGGGCTTCTCCTCCGGGCTCGTACG - ίο A tandem construction of the synthetic and the tendamistat promoter is thereby obtained. The plasmid is called pGRUO.

If the synthetic fragments (1), (2) and (3) are, after 5 cutting with SphI and Sstl, inserted into pGRUO, the expression vectors pGR200, 210 and 220 result. In an analogous way, the expression vectors pGR250, 260 and 270 are obtained with fragment (4).

Example 8 If it is intended to produce human insulin from the insulin precursors by combining trypsin, or an enzyme with an identical effect, and carboxypeptidase B, it is advantageous to cleave off rapidly the ballast portion in the course of the cleavage reaction in order to favor the cleavage reaction leading to the B31 (Arg)-insulin. For this purpose, a modification of the amino acids upstream of amino acid BI (Phe) is suitable: The procedure is analogous to Example 1 and the plasmid pKK 500 is opened using the restriction enzymes EcoRI and Drain. The original fragment is then replaced by DNA fragment (6) (SEQ ID NO:6) BI Asn Ser Ala Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu 5' AAT TCG GCC CGC TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC CTC 3' 3' GC CGG GCG AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG 5' (EcoRI) (Dralll) which has been synthesized by the phosphoramidite method. Cloning into E. coli and expression in Streptomyces lividans are carried out in accordance with Example 1 and Example 2, respectively. Plasmid pKK640 and expression plasmid pKF14 result. - 11 The plasmid which results according to Example 5 (after incorporation of fragment (4)) can be treated in an analogous way. The plasmids pKK650 and pKF15 are obtained in this way.

Annex ( from EP-A . 0,367,: 163) : Table 1 B1 10 ASN SER ASN GLY LYS PHE VAL ASN GLN HIS LEU CYS GLY SER HIS AAT TCG AAC GGC AAG TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC GC TTG CCG TTC AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG (EcoRI) 20 30 LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARC GLY PHE PHE CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG TYR THR PRO LYS THR C LYS A1 GLY ILE VAL 40 GLU GLN CYS CYS THR SER TAC ACC CCC AAG ACC AAG GGC ATC GTG GAG CAG TGC TGT ACG TCC ATG TGG GGG TTC TGG TTC CCG TAG CAC CTC GTC ACG ACA TGC AGG ILE CYS SER LEU 50 TYR GLN LEU GLU ASN TYR CYS ASN STP STP ATC TGC TCC CTC TAC CAG CTC GAG AAC TAC TGC AAC TAG TAA TAG ACG AGG GAG ATG GTC GAG CTC TTG ATG ACG TTG ATC ATT GTC GAC CTG CAG CCA • CAG CTG GAC GTC GGT TCG A Sail (Hindlll) Table 2 ’ - CG ATAAACCDATACAATTAAAGGCTCCTTITGGAGCCITnTTTTTGGAGATTTTCAACGTGGATC GCTATTTGGCrATGnAATTrCOGAGGAAAACCTOGGAAAAAAAAACCTCTAAAAGTTGCACCTAG-5

Claims (12)

1. Patent claims:

1. A process for the production of fusion proteins, which comprises coupling the structural gene for the desired protein to the codons for the signal sequence and about 5 the first ten amino-terminal amino acids of tendamistat, expressing this genetic structure in a streptomyces host cell and isolating the secreted fusion protein from the supernatant, it being possible for the tendamistat gene sequences to be modified. 10

2. . A genetic structure containing the signal sequence and about the first ten codons for tendamistat and the structural gene for another protein.

3. The genetic structure as claimed in claim 2, wherein codons for amino acids have been deleted, exchanged or 15 added in the tendamistat portion.

4. The genetic structure as claimed in claim 2 or 3, wherein a linker sequence is arranged between the tendamistat gene and the structural gene for the desired protein. 20

5. A vector containing a genetic structure as claimed in claim 2, 3 or 4.

6. A streptomyces cell containing a vector as claimed in claim 5.

7. A fusion protein, wherein an N-terminal portion of 25 about the first ten amino acids of tendamistat is coupled, if appropriate via a bridge sequence, to the desired protein.

8. The use of the fusion protein as claimed in claim 7 or of the fusion protein obtainable as claimed in claim 1 30 for the production of the desired protein.

9. A process as claimed in claim 1, substantially as hereinbefore described and exemplified.

10. Fusion proteins whenever produced by a process claimed in claim 1 or 9.

11. A genetic structure as claimed in claim 2, substantially as hereinbefore described and exemplified.

12. A fusion protein as claimed in claim 7, substantially as hereinbefore described and exemplified.