IE62522B1 - A process for the preparation of foreign proteins in streptomycetes - Google Patents

Abstract

The tendamistat gene can be used for the construction of gene fusions with which fusion proteins can be expressed and excreted in Streptomycetes host cells. The tendamistat portion can be modified, in particular truncated at the C-terminal end.

Description

The European patent application with the publication number (EP-A) 0,161,629, and South African Patent 85/3672 disclose the use of the DNA coding for the signal peptide (prepeptide) of the α-amylase inhibitor tendamistat in order for a Streptomyces cell to excrete a polypeptide, in particular tendamistat. The appropriate DNA can, in principle, be obtained from every strain producing tendamistat, but a DNA obtained as in Example 3 of German Offenlegungsschrift 3,331,860 is preferably used.

Patent application 88103141.3 (corresponding to German patent application P 37 07 150.5 of March 6, 1987) has already proposed a process for the excretion of fusion proteins from Streptomyces, which comprises incorporating the coding sequence for the desired protein in the tendamistat gene, which has been modified where appropriate, and expressing the recombinant gene in a Streptomyces cell. Thus, in this case the tendamistat structural gene is used as a carrier for another gene, the fusion proteins which are obtained having the amino acid sequence of another protein located within the tendamistat amino acid sequence. Consequently, on chemical or enzymatic cleavage of this fusion protein to liberate the other protein, two tendamistat partsequences are obtained. The said earlier application also relates to tendamistat derivatives, which are understood to include those with a markedly truncated amino acid chain. Derivatives of this type are able in a reversible manner to react with the specific receptors in the form of a competitive inhibitory mechanism.

It has now been found that foreign proteins can also be prepared in Streptomyces by constructing fusion protein genes in which there is C-terminal coupling of the structural gene for the desired protein to the tendamistat gene, which has been modified where appropriate. The modification of the tendamistat gene may comprise, in particular, C-terminal truncation.

The DNA coding for tendamistat is depicted in EP-A 0,161,629 (where it is DNA sequence C; Table 1 in the annex). This structural gene contains several cleavage sites for restriction enzymes, which can be used to modify the coded amino acid sequence. Suitable cleavage sites are those for BstEII 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. It is possible, by incorporation of appropriate linkers, to insert at these sites one or more additional amino acids, to eliminate DNA segments between these cleavage sites, or to code for truncated amino acid sequences by incorporation of stop codons. Furthermore, it is possible by sitespecific mutagenesis for any desired amino acids to be inserted, replaced or eliminated. In this way proteins are obtained which have an α-amylase inhibitory action, as well as proteins which do not have this activity but still react with the corresponding receptors.

The invention also relates to appropriate gene structures, vectors containing these gene structures, Streptomyces cells transformed with these vectors, the excreted fusion proteins, and their use for the preparation of the foreign proteins and tendamistat derivatives. Preferred embodiments of the invention are explained in detail hereinafter and defined in the patent claims.

Figures 1 to 4 depict some plasmid constructions according to the invention.

Fig. 1 shows the preparation of the hybrid plasmid pKK310 which codes for a fusion protein in which part of the tendamistat amino acid sequence is followed by a bridging member of seven amino acids and, thereafter, the amino acid sequence of monkey proinsulin.

Fig. 2 shows the transfer of the plasmid pKK310 into the expression plasmid pTFl.

Fig. 3 shows the construction of the plasmid pRSlO in which part of the tendamistat gene is followed by the polylinker from pUC18, and its transfer into the expression plasmid pTFlO. mcs denotes the polylinker region (multiple cloning site) of pUC18.

Finally, Fig. 4 shows the construction of the plasmid pKK400 which codes for a fusion protein in which the whole of the amino acid sequence of tendamistat is followed by a bridging member of eleven amino acids and, thereafter, the amino acid sequence of monkey proinsulin, and its transfer into the expression plasmid pGFl.

The figures are not drawn true to scale.

The fusion proteins obtained according to the invention, which are exported from the cell, have the advantage that they can readily be isolated from the culture filtrate. The isolation can be carried out in a manner known per se, advantageously by adsorption or ion exchange chromatography and/or gel filtration.

The desired foreign protein (fusion partner) is liberated by enzymatic or chemical cleavage likewise in a manner known per se.

In this connection, the type' of cleavage depends, in particular, on the amino acid sequence of the desired protein. It will be expedient in many cases to incor25 porate a connecting member or bridging member in the cleavage site between the tendamistat sequence and the amino acid sequence of the desired protein. If the desired protein contains, for example, no methionine, the connecting member can denote Met, whereupon chemical cleavage with cyanogen chloride or bromide is carried out. If the connecting member has a carboxyl-terminal cysteine, or if the connecting member represents Cys, it is possible for enzymatic cysteine-specific cleavage, or ρΗρτη-ί nal cleavage, for example after specific S-cyanylation, to follow. If the bridging member has a carboxyl-terminal tryptophan, or if the connecting member represents Trp, chemical cleavage with N-bromosuccinimide can be carried out.

Desired proteins which do not contain Asp-Pro in their amino acid seguence and are sufficiently stable to acid can, as fusion proteins having this bridging member, be cleaved proteolytically in a manner known per se. This results in proteins which contain N-terminal proline or C-terminal aspartic acid. Thus, it is also possible in this way to synthesize modified proteins.

The Asp-Pro bond can be made even more labile to acid if this bridging member denotes (Asp)n-Pro or is Glu- (Asp) Pro, n denoting 1 to 3.

Examples of enzymatic cleavages have also been disclosed, it also being possible to use modified enzymes having improved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, it is expedient to choose a peptide sequence in which an amino acid which can be eliminated by trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of the proinsulin, for example Ala-Ser-Met- [lacuna] If the desired protein does dot contain the amino acid sequence Ile-Glu-Gly-Arg, the fusion protein having the corresponding bridging member can be cleaved with factor Xa (EP-A 0,025,190 and 0,161,973).

The isolation of the cleavage products depends on the properties of these proteins. Concerning the isolation of tendamistat and its derivatives, reference may be made to the literature cited in German Of fenlegungsschrift 3,331,860.

The invention is explained in detail in the examples which follow. Unless otherwise indicated, percentage data relate to weight.

The examples are illustrated by figures with the same number. The plasmids and gene fragments are not depicted to scale in the figures; in particular the scale is stretched in polylinker sequences.

Example 1 The starting material used is the plasmid pKAI650, which is described in German „O.ffenlegungsschrift 3,536,182 and in EP-A 0,218,204. This plasmid can be obtained from the plasmid pKAIl, which is described in German Of fenlegungsschrift 3,331,860, by isolation of the 650 bp HincII/Sstl fragment and cloning into the plasmid pUC19 which has been opened with these enzymes. The unique Hindlll cleavage site in this plasmid is removed (by cutting with this enzyme, filling in the protruding ends, and ligation) to result in the plasmid pKAI650a (1). gg of (1) DNA purified by CsCl gradient centrifugation are completely digested, in a 50 gl reaction mixture, with Stul for 2 hours as stated by the manufacturer, and the enzyme is removed by phenol extraction. The linearized DNA is precipitated with* ethanol, redissolved and introduced into a ligation mixture to which is added, as additional reactant, 0.1 gg of the chemically synthesized double-stranded oligonucleotide (2) which has been phosphorylated at the 5' end Hindlll ' CCCAAGCTTGGG 3' (2) 3' GGGTTCGAACCC 5' Transformation of the ligation mixture into E. coli UM 109 is followed by isolation of those clones which harbor the recombinant plasmid pKK3a (3) . The isolated plasmid DNA has a cleavage site for the restriction enzyme Hindlll, which permits characterization by restriction analysis. pKK3a (3) is 12 base-pairs larger than pKAI650a (1) and has a nucleotide sequence which extends the amino acid sequence by 4 amino acids, and follows 43 (44) Glu Gly Pro Ser Leu Gly Leu 5' GAA GG C CCA AGC TTG GG C CTG 3' 3' CTT CC G GGT TCG AAC CC G GAC 5' Hindlll Another starting material used is the plasmid pYE24 (4). This plasmid is obtained by opening the vector pUC8 with EcoRI and Hindlll, and ligating into this linearized plasmid the gene for monkey proinsulin (Table 2; cf. Wetekam et al., Gene 19 (1982) 179-183). ^g of the plasmid pYE24 (4) are reacted with the restriction enzymes EcoRI and Hindlll, and the gene for monkey proinsulin is isolated by electroelution and, after purification and concentration by ethanol precipitation, it is ligated with the synthetic DMA linker (5) ' AGC TTG ATG GCG 3' 3' AC TAC CGC TTA A 5' (5) (Hindlll) (EcoRI) The ligation product (6) is now inserted into the plasmid pKK3a (3) which has been opened with Hindlll, resulting in the plasmid pKK31 (7) . This construction results in the following bridging member being downstream of the codon for amino acid Gly 43 of the tendamistat gene: Β 1 Gly Pro Ser Leu Met Ala Asn Ser Phe GGC CCA AGC TTG ATG GCG AAT TCT TTT CCG GGT TCG AAC TAC CGC TTA AGA AAA Hindlll EcoRI Ί Β 1 here, and in Table 2, designates the start of the B chain of monkey proinsulin.

In plasmid (7) the proinsulin sequence is located within the tendamistat gene. To transfer this plasmid into a plasmid according to the invention, (7) is digested with SphI and Sail, and the fragment (8) is isolated. The vector pUC19 is opened with SphI and Sail, and the linearized plasmid is ligated with the fragment (8). The resulting plasmid pKK310 (9) codes for a fusion protein in which the truncated tendamistat sequence and the linker which is depicted above are followed only by the proinsulin sequence.

The entire construction is depicted in Figure 1.

Example 2 To transfer the plasmid pKK310 (9) in an expression plasmid, (9) is reacted with Sstl and SphI, and the fragment (10) is isolated.

The commercially available expression vector pIJ702 (11) (obtainable from the John Innes Foundation, Norwich, England) is opened with SphI and Sstl, and the linearized plasmid (12) is ligated with the fragment (10) . After transformation of the strain Streptomyces lividans TK 24 (John Innes Foundation), the desired clones are identified by selection for resistance to thiostrepton. The plasmid DNA from thiostrepton-resistant clones is isolated and examined by restriction analysis. Plasmids having the desired orientation of the gene are called pTFl (13). Clones which contain this recombinant plasmid secrete a protein of molecular weight 16 kD into the culture medium. This protein shows a positive immunoblotting reaction with insulin antibodies (cf. Example 5).

The construction of pTFl (13) is depicted in Figure 2.

Example 3 The plasmid pKK3a (3), on the one hand, and the vector pUC18, on the other hand, are each opened with Hindlll, and are ligated together. The ligation mixture is used to transform the E. coli strain JM 109, which indicates successful cloning in the presence of isopropyl-β-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyld-D-galactopyranoside (X-Gal) by the formation of colorless colonies. The resultant recombinant plasmid pRSl (14) is isolated in a manner known per se. Digestion of 1 pg of the plasmid with the restriction enzyme Sstl, followed by relegation- results in deletion of the pUC18 portion apart from the polylinker sequence (mcs) and the remainder of the tendamistat gene. The plasmid pRSlO (15) is obtained.

The plasmid (15) is, owing to its polylinker portion, suitable for cloning any desired structural genes, resulting in plasmids which code for the corresponding fusion proteins with the truncated tendamistat sequence.

When pRSlO (15) is digested with SphI and Sstl, and the smaller fragment is isolated, the latter can be ligated into the expression vector pIJ702 in analogy to Example 2. In this way the expression vector pTFlO (16) is obtained, and this likewise, by reason of its polylinker portion, allows versatile constructions.

The construction of pTFlO (16) is depicted in Figure 3.

Exanple 4 The plasmid pYE24 (4) is opened with EcoRI, and the linker ' AAT TCA AGC TTG 3' GT TCG AAC TTA A (EcoRI) Hind III (EcoRI) is inserted, resulting in the plasmid pYE241. Cutting with Hindlll, and ligation into pKK3a (3) cut with Hindlll results in the plasmid pKK32, in analogy to Example 1. The latter codes for a fusion protein in which the tendamistat sequence is linked to the proinsulin sequence by the following bridging member: 43 Gly Pro Ser Leu Asn Phe Ala Arg GGC CCA AGC TTG AAT TCT GCA AGA TTT CCG GGT TCG AAC TTA AGA CGT TCT AAA In analogy to Example 1, pKK32 is cut with SphI and Sstl, and the fragment which is approximately 650 bp in size is cloned into pUC19, which has been opened with these enzymes. The resulting plasmid pKK320 corresponds to a plasmid pKK310 (9) apart from the abovementioned bridging member (in which the sequence introduced by the linker is emphasized by emboldening).

In analogy to Example 2, the Sstl-SphI fragment having the recombinant gene from pKK320 is cloned into pIJ702, resulting in the expression plasmid pTF2. A fusion protein of 16 kd is expressed and secreted in S. lividans TK 24, and the protein reacts with insulin antibodies (cf. Example 5).

Because of the similarity of the construction of pTF2 to that of pTFl (13), Figures 1 and 2, no depiction in a drawing has been given.

Example 5 pKK310 (9) is partially digested with EcoRI so that only one of the two EcoRI cleavage sites is opened. After the protruding ends have been filled in using Klenow polymerase, the plasmid is religated, and the result is checked by restriction analysis. The desired plasmid, in which the EcoRI site located at the end of the proinsulin gene has been eliminated, is called pKK310a (17) . Thus, the latter now contains a unique restriction site for EcoRI in the linker region between the truncated tendamistat gene and the proinsulin gene.

To construct the plasmid which codes for a fusion protein having the complete tendamistat sequence, a unique cleavage site for Kpnl is introduced, in the region of the codons for amino acids 68/69, into the DNA sequence coding for tendamistat (Table 1) . This entails the isolated DNA from pKAI650a (1) being digested with Sst and Sphl, and the fragment which is 650 bp in size being cloned into the phage M13mpl8 RF DNA, which has .likewise been digested with these two enzymes, and the singlestranded DNA being prepared by known methods. 1 /xg of this ssDNA is used together with 0.1 /xm of the mutagenic primer · C GAG GTA CCG GGC GT 3' in site-directed mutagenesis (M.J. Zoller and J. Smith, Nucleic Acid Res. 10 (1982) 6487-6500).

The RF DNA is isolated from the isolated M13 clones having the mutated gene, which can be selected by the additional Kpnl cleavage site, and the base exchange (C for G at the third position *in the codon for Arg68) is confirmed by sequencing. Thus, the nucleotide exchange brings about no change in the amino acid sequence but does introduce the desired new unique cleavage site into the tendamistat structural gene. 66 67 68 69 70 His Ala Arcr Tvr Leu • CAC GCC CGC TAC CTC GTC CGG GCC ATC GAG Kpnl The mutated sequence is, after Sstl-SphI digestion, cloned out of the M13mpl8 RF DNA into the plasmid pUC19, resulting in the plasmid pKAI651 (18).

To check, the 650 bp SStl-SphI insert from (18) is incorporated, as in Example 2, into the plasmid pIJ702, resulting in the plasmid pAX651. After this plasmid has been transformed into Streptomyces lividans TK 24, the expression rates for tendamistat which are obtained are the same as for the plasmid pAX650 having the unmoHj f j ed tendamistat gene (German Offenlegungsschrift 3,536,182, Fig. 3).

To prepare a plasmid, according to the invention, for a fusion protein having the entire amino acid sequence of tendamistat, the plasmid pKAI651 (18) is now digested with SphI and Kpnl, and the small fragment is ligated with the linker (19) 69 70 71 72 73 74 (Tyr) Leu Ala Arg Cys Leu Phe Asn Ala Met Ala Thr Gly 5' CTC GCT CGC TGC CTT TTC AAT GCG ATG GCC ACC GGG 3' ATG GAG CGA GCG ACG GAA AAG TTA CGC TAC CGG TGG CCC TTA A 5' (Kpnl) (19) (EcoRI) and the plasmid pKK310a (17) which has been opened with SphI and EcoRI.

The ligation mixture is used to transform E. coli JM 109, the plasmid DNA is isolated, and the correct fusion is verified by DNA sequencing. The plasmid having the correct sequence is called pKK400 (20).

The linker (19) codes not only for the remaining amino acids of tendamistat but also for the portion of a spacer which separates the tendamistat - and the proinsulin gene - from one another, and overall embraces - with the 5' end of the gene as shown in Table 2 - the codons for the following 11 amino acids: Phe-Asn-Ala-Met-Ala-Thr-Gly-Asn-Ser-Ala-Arg Thus, the fusion protein contains in this spacer, inter alia, the amino acids methionine and arginine, which permit cleavage with cyanogen halide or trypsin.

The insert of about 1090 bp is isolated from the plasmid pKK400 (20) by double-digestion with Sstl and SphI, and the DNA is ligated into the plasmid pIJ702 (12) which has been opened with the same enzymes. The result is the plasmid pGFl (21). The ligation mixture is transformed into S. lividans TK 24, and the plasmid DNA is isolated from thiostrepton-resistant transformants, which have tendamistat activity. All positive clones contain the pGFl Sstl-SphI insert which is 1090 bp in size.

The construction of pGFl (21) is depicted in Figure 4.

The tendamistat activity is determined by the plate assay which is described in Example 3 in EP-A1 0,161,629 and in Example 2 in German Offenlegungsschrift 3,536,182.

The fusion protein coded for by pGFl can be expressed in a known manner. When the transformed strain S. lividans TK 24 is incubated in shaken flasks at 28°C for 4 days, and the mycelium is removed from the culture solution by centrifugation, the fusion protein can be detected in the clear solution as follows: to 100 μΐ of solution are mixed with 20 to 200 μΐ of 15% strength trichloroacetic acid, and the precipitated protein is concentrated by centrifugation, washed and taken up in SDS-containing sample buffer (U. Laemmli, Nature 227 (1970) 680-685) . After incubation at 90°C for 2 minutes electrophoresis fractionation on a 10 to 17% strength SDS polyacrylamide gel is carried out. A protein of molecular weight 19 kD is obtained, that is to say in the expected molecular weight range for the fusion protein composed of tendamistat and proinsulin. The - 13 fusion protein reacts both with antibodies against tendamistat and with antibodies against insulin.

Claims (14)

1. Patent Claims:

1. A process for the preparation of fusion proteins, which comprises coupling of the structural gene for the desired protein onto the 3' end (of the coding strand) of the tendamistat gene, which is modified where appropriate, bringing about the expression of this gene structure in a Streptomyces host cell, and isolating the secreted fusion protein from the supernatant.

2. The process as claimed in claim 1, wherein the tendamistat gene is truncated at the 3' end.

3. A gene structure containing the tendamistat gene, which is modified where appropriate, onto the 3' end of which a structural gene for another protein has been coupled.

4. A gene structure as claimed in claim 3, wherein the tendamistat gene is truncated at the 3' end.

5. A vector containing a gene structure as claimed in claim 3 or 4.

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

7. A fusion protein which has only one N-terminal portion of tendamistat, which is modified where appropriate.

8. The use of the fusion protein as claimed in claim 7, or of the fusion protein obtainable as claimed in claim 1 or 2, for the preparation of tendamistat, which is modified where appropriate, and/or of the fusion partner.

9. A process according to Claim 1 for the preparation of a fusion protein, substantially as hereinbefore described and exemplified.

10. A fusion protein whenever prepared by a process 5 claimed in any one of Claims 1, 2 or 9.

11. A gene structure according to Claim 3, substantially as hereinbefore described and exemplified.

12. A vector according to Claim 5, substantially as hereinbefore described and exemplified. 10

13. A fusion protein according to Claim 7, substantially as hereinbefore described and exemplified.

14. Use according to Claim 8, substantially as hereinbefore described and exemplified.