FI97549B - Process for the preparation of foreign proteins in Streptomyces cells - Google Patents

Description

97549

Method for producing foreign proteins in Streptomyces cells Use of DNA encoding the α-amylase inhibitor tendamate signal peptide (prepeptide) for secretion of a polypeptide, in particular tendamate, in Streptomyces cells is known from EP patent application. ZA Patent Publication 85/3672. The DNA in question can in principle be obtained from any tendamate-10-producing strain, but preferably the DNA obtained according to Example 3 of DE-A-3 331 860 is used.

FI Patent Application No. 880,986 has previously disclosed a method for secreting foreign proteins from Strepto-15 myces cells, which was characterized by introducing the sequence encoding the desired protein into a possibly modified tendamat gene and expressing the recombinant gene in the Streptomyces cell. Thus, the tendamatt structural gene is used as a carrier for another gene and fusion-20 proteins are obtained in which the amino acid sequence of the second protein is arranged in the middle of the tendonate amino acid sequence. Cleavage of this fusion protein chemically or enzymatically to release the foreign protein thus yields two tendamate subsequences. Said earlier patent application also relates to tendamate derivatives, which means e.g. also derivatives comprising a substantially truncated amino acid residue chain. These derivatives are capable of reacting reversibly with specific receptors in a competitive inhibitory reaction.

We have since found that foreign proteins can also be produced in Streptomyces cells by constructing fusion protein genes in which the structural gene of the desired protein is linked to the 3 'end of a potentially modified tendamate gene (coding strand). In particular, modification of the tendamate-35 gene may involve truncation of the chain from the 3 'end.

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The DNA encoding tendamate is disclosed in EP-A 0 161 629 (where it is shown as DNA sequence C and in Table 1 in the Annex). This structural gene comprises a number of restriction enzyme centers suitable for use in modifying the amino acid sequences to be encoded. Suitable sites are BstEII restriction centers in the region of triplets 31 and 32, Stul centers in the region of triplets 43 and 44, and Sau3A centers in the region of triplets 52 and 53. With the help of suitable linkers, one or more additional amino acid residues can be placed in these centers, or DNA segments between these restriction centers can be removed or truncated amino acid sequences encoded by the addition of additional termination codons. In addition, site-directed mutation 15 can be used to add, change, or remove specific amino acid residues. This results in proteins that have α-Amylase inhibitory activity, or proteins that lack this activity but still react with the corresponding receptors.

The invention further relates to corresponding gene constructs, vectors containing these gene constructs, Streptomyces cells transformed with these vectors, secreted fusion proteins, and their use for the production of foreign proteins and tendamate derivatives. In the following, the preferred embodiments of the invention, which are likewise defined in the appended claims, will be explained in more detail.

Figures 1-4 show some plasmid structures according to the invention.

Figure 1 shows how we prepared the hybrid-30 plasmid pKK310, which encodes a fusion protein in which part of the tendonate amino acid sequence is followed by a bridging sequence of seven amino acid residues, followed by the amino acid sequence of insulin monkey insulin.

Figure 2 shows the transfer of plasmid pKK310 to expression plasmid pTF1.

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Figure 3 shows how we constructed plasmid pRS10, in which part of the tendamate gene is followed by a polylinker derived from pUC18, and how the plasmid was transferred into the expression plasmid pTF10. The term "mcs" refers to the multiple cloning site of pUC18.

Finally, Figure 4 shows how we constructed plasmid pKK400, which encodes a fusion protein in which the complete amino acid sequence of tendamate is followed by a bridging sequence of 11 10 amino acid residues, followed by the amino acid sequence of monkey insulin insulin, and how the plasmid was transferred into the expression plasmid.

The patterns are not drawn on a special scale.

The cell-derived fusion proteins obtained according to the invention are preferred in that they can be easily isolated from the culture filtrate. Isolation can take place in a manner known per se, preferably using adsorption or ion exchange chromatography and / or gel filtration.

The release of the desired foreign protein (fusion partner) by enzymatic or chemical cleavage is likewise carried out in a manner known per se.

In this case, the method of cleavage is determined primarily by the amino acid sequence of the desired protein. In several cases, it is convenient to insert a splice sequence, i.e., a bridge sequence, into the restriction center between the tendamate sequence and the amino acid sequence of the desired protein. For example, if the desired protein does not contain methionine, Met-ice-30 can be used as the linker, in which case the cleavage to be performed is carried out chemically with chlorine or bromocyano. If the linker sequence comprises a cysteine residue at its carboxyl terminus, or if the linker sequence consists of a Cys residue, enzymatic cysteine-specific cleavage or, for example, chemical cleavage following specific S-cyanylation may be used in the cleavage to be performed. When the bridge sequence comprises a tryptophan residue at its carboxyl terminus or is formed from a Trp residue, chemical cleavage can be performed with N-bromosuccinimide.

The desired proteins which do not contain the Asp-Pro sequence in their amino acid sequence and are in addition sufficiently acid stable can be proteolytically cleaved from fusion proteins comprising this bridging sequence in a manner known per se. In this case, proteins are obtained which have a proline at the N-terminus or an aspartic acid residue at the C-terminus. Thus, modified proteins can also be synthesized.

The Asp-Pro bond can be made even more easily cleaved by acid using the structure (Asp) -Pro 15 or Glu- (Asp) n ~ Pro as the bridge sequence, where n = 1 to 3.

Examples of enzymatic cleavage are also known, in which modified enzymes with better specificity can also be used (cf. C.S. Cr. Et al., Science 228 (1985) 291-297). When the desired eukaryotic peptide is proinsulin, it is appropriate to select a peptide sequence in which a trypsin-cleavable amino acid residue (Arg, Lys) is attached to the N-terminal amino acid residue (Phe) of proinsulin, for example, the sequence Ala-Ser-Met-.

25 Unless the desired protein contains an amino acid sequence

Ile-Glu-Gly-Arg, a fusion protein containing said sequence as a bridge sequence can be cleaved by factor Xa (EP-A 0 025 190 and 0 161 973).

The method of isolation of the cleavage products is determined by the properties of the proteins in question. With regard to the isolation of tendamate and its derivatives, we refer to the literature referenced in DE-Application-35 3 331 860.

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The following examples illustrate the invention in more detail. Unless otherwise stated, percentages are by weight.

The examples are illustrated by an image with the corresponding sequence number-5. In the figures, plasmids and gene fragments are not shown on a scale, in particular the scaler has been scaled up in particular for polyether linker sequences.

Example 1 The plasmid pKAI650 described in DE-A-3 536 182 and EP-A-0 218 204 is used as a starting material. This plasmid can be obtained by isolating a 650 bp HincII-SstI fragment of pKAI1 from the plasmid described in DE-A-3 331 860. and cloning it into plasmid pUC19 opened with the aforementioned enzymes. Individual HindIII restriction centers are removed from the plasmid (by digestion with the enzyme in question, filled in with end extensions and ligated) to give plasmid pKAI650a (1).

20 [mu] g of plasmid (1) DNA purified by CsCl gradient centrifugation are digested completely for 2 hours with a reaction addition of 50 [mu] l according to the manufacturer's instructions and the enzyme is removed by phenol extraction. Precipitate the linearized DNA with ethanol, redissolve it, and then transfer it to a ligation mixture to which 0.1 μg of chemically synthesized, 5'-terminal phosphorylated double-stranded oligonucleotide (2) is added as a second reactant.

Hindlll 30 5 'C C C A A G C T T G G G 3' (2) 3 'GGGTTCGAACCC 5'

E. coli strain JM109 is transformed with the ligation mixture and clones containing the recombinant plasmid pKK3a (3) are isolated. The isolated plasmid DNA is characterized by the presence of the restriction enzyme HindIII center, which may. 97549 6 revives identification by restriction enzymatic analysis. pKK3a (3) is 12 base pairs larger than pKA166 (1) and has a nucleotide sequence that elongates the amino acid sequence by 4 amino acid residues 5 as follows: 42 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'

Hind III

Plasmid pYE24 (4) is used as a second starting material.

This plasmid is obtained by opening the vector pUC18 with EcoRI and HindIII and ligating the monkey insulin insulin gene into the resulting linearized plasmid (Table 2; cf. Wetekam et al., Gene 19 (1982) 179-183).

2 ug of plasmid pYE24 (4) is digested with the restriction enzymes EcoRI and HindIII, isolated from the monkey insulin gene by electroelution and ligated after purification and concentration by ethanol precipitation after synthesis of the synthetic DNA linker 25 5 'AGC TTG ATG GCG 3' 3 'AC TAC CGC TTA A 5 '(5) (HindIII) (EcoRI) 30. The ligation product (6) is then introduced into the HindIII-opened plasmid pKK3a (3) to give plasmid pKK31 (7). This construct generates the following bridge sequence associated with codon Gly 43 of the amino acid residue of the ten-damistat gene:

II

•, - 97549 7 43 B 1

Gly Pro Ser Leu Met Ala Asn Ser Fhe

GGC CCA AGC TTG ATG GCG AAT TCT TTT 5 CCG GGT TCG AAC TAC CGC TTA AGA AAA

HindiII EcoRI

B 1 here, as in Table 2, denotes the origin of the B chain of monkey proinsulin.

10 In plasmid (7), the proinsulin sequence is located in the tendamate gene. To convert this plasmid to the plasmid of the invention, plasmid (7) is cleaved

With SphI and SalI and fragment (8) is isolated. Plasmid pUC19 is opened with SphI and SalI and the linearized plasmid is ligated to fragment (8). Plasmid pKK310 (9) thus obtained encodes a fusion protein in which the truncated tenamate sequence and the above linker are further followed by the proinsulin sequence.

The construction as a whole is shown in Figure 1.

Example 2

To transfer plasmid pKK310 (9) to the expression plasmid, plasmid (9) is digested with SstI and SphI and fragment (10) is isolated.

The commercially available expression vector pIJ702 (11) (available from the John Innes Foundation, Norwich, England) is opened with Sphlt and SstI and the linearized plasmid (12) is ligated to fragment (10). Strain Streptomyces lividans TK 24 (John Innes Foundation) is transformed to identify the desired clones by selection for thiostrepton resistance. Plasmid DNA is isolated from thiostrepton-resistant clones and confirmed by restriction enzyme analysis. Plasmids containing the gene in the desired orientation are designated 35 pTF1 (13). Clones containing this recombinant plasmid secrete a protein with a molecular weight of 8,97549 16 kD into the medium. This protein reacted positively with insulin antibodies in a blot immunoassay. (See Example 5).

The construction of pTF1 (13) is shown in Figure 2.

5 Example 3

Plasmid pKK3a (3) and vector pUC18 are opened separately with HindIII and ligated together. Transformation with the ligation reaction product E. coli strain JM109 showing successful cloning of isopropyl-8-thiogalactopyranos-10 din (IPTG) and 5-bromo-4-chloro-3-indolyl-8-D-galactopyranoside (X-Gal in the presence of colorless colonies. The resulting recombinant plasmid pRS1 (14) is isolated in a manner known per se. Cleavage of 1 μg of plasmid with the restriction enzyme SstI followed by re-ligation deletes the pUC18 portion up to the polylinker sequence (mcs) and the remainder of the tendamate gene. Plasmid pRS10 (15) is obtained.

Plasmid (15) is suitable for cloning various structural genes due to its polyether linker to give plasmids encoding 20 corresponding fusion proteins with a truncated tendamate sequence.

If pRS10 (15) is cleaved with SphI and SstI and less of the resulting fragments are isolated, this can be ligated analogously to Example 2 to the expression vector pIJ702. This gives the expression vector pTFIO (16), which also allows the creation of several different structures due to its poly-linker moiety.

The construction of pTFIO (16) is shown in Figure 3.

Example 4 Plasmid pYE24 (4) is opened with EcoRI, the linker 5 'AAT TCA AGC TTG 3' 3 'GT TCG AAC TTA A 5' 35 (EcoRI) Hind III (EcoRI) 9 97549 is added and plasmid pYE241 is obtained. Cleavage with HindIII and ligation into the HindIII-cut plasmid pKK3a (3) gives plasmid pKK32 analogously to Example 1.

This encodes a fusion protein in which tendamate sequence 5 is linked to the proinsulin sequence via the following bridging sequence: 43

Gly Pro Ser Leu Äsn Phe Ala Arg

GGC CCA AGC TTG AAT TCT GCA AGA TTT

10

CCG GGT TCG AAC TTA AGA CCT TCT AAA

As in Example 1, plasmid pKK32 is digested with SphI and SstI and the resulting fragment of about 650 bp is subcloned into the previously opened pUC19 by said enzymes. The resulting plasmid pKK320 corresponds to plasmid pKK310 (9) up to the bridging sequence shown above (where the linker-inserted sequence is indicated in bold).

In analogy to Example 2, the SstI-SphI fragment plasmid pKK320 containing 20 recombinant genes is subcloned into plasmid pIJ702 to give the expression plasmid pTF2. Strain S.lividans TK24 expresses and secretes a 16 kD fusion protein that reacts with insulin antibodies (cf. Example 5).

With the construction of pTF2 substantially similar to that of pTF1 (13), Figures 1 and 2, one illustrative representation was satisfied.

Example 5

Plasmid pKK310 (9) is partially digested with EcoRI so that only one of the two EcoRI centers is cleaved open. End extensions are filled in with Klenow polymerase, after which the plasmid is re-ligated and the structure of the products is confirmed by restriction analysis. The desired plasmid in which the EcoRI center located at the end of the proinsulin gene has been eliminated is designated plasmid pKK310a (17). Thus, it now contains only one EcoRI-10 97549 restriction site located in the fusion region between the truncated tendamism gene and the proinsulin gene.

To construct a plasmid encoding a fusion protein containing the complete 5 tendamate sequences, a single KpnI center is introduced into the codon region of amino acid residues 68/69 of the DNA sequence encoding the tendonate (Table 1). To this end, the isolated pKAI650a (1) DNA is digested with SstI and SphI and a 650 bp oral fragment is subcloned into the circulating DNA of M13mp18 phage, which was also digested with these enzymes, and Single-stranded DNA is prepared: according to known methods. 1 ug of this single strand DNA and 0.1 ug of mutation primer are used.

15'C GAG GTA CCG GGC GT 3 'in site-directed mutation (M.J. Zoller and J.Smith, Nucleic Acid Res. 10 (1982) 6487-6500).

Isolated M13 clones containing the mutant gene, which are preselected by the added KpnI center, isolate ring DNA and confirm base exchange (C changes to G at position 3 of the Arg codon). Thus, the nucleotide exchange does not cause any change in the amino acid sequence, however, allowing the desired one new restriction center to be introduced into the tendamate structural gene.

66 67 68 69 70

His Ala Arg Tyr Leu 30 CAC GCC CGC TAC CTC

GTC CGG GCC ATC GAG

KpnI

The mutated sequence is subcloned by SstI-SphI digestion from M13mp18 ring DNA into plasmid pUC19 to give plasmid pKAI651 (18).

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As a control, the 650 bp SstI-SphI insert of plasmid (18) is transferred to plasmid pIJ702 according to the procedure of Example 2 to give plasmid pAX651. Transformation of Streptomyces lividans strain TK 5 24 with this plasmid gives the tendonate the same expression frequency as plasmid pAX650 containing the original tendonate gene (DE-A-3,536,182, Figure 3).

To prepare the plasmid of the invention with respect to the fusion protein comprising the entire amino acid sequence of ten-damistat, plasmid pKAI651 (18) is then digested with SphI and KpnI and the resulting smaller fragment ligated together, linker (19) 69 70 71 72 73 74 15 (Tyr) Leu Le Arg Cys Leu Phe Asn Ala Met Ala Thr Gly 3 '

5 'CTC GCT CGC TGC CTT TTC AAT GCG ATG GCC ACC GGG

3 'C ATG GAG CGA GCG ACG GAA AAG TTA CGC TAC CGG TGG CCC TTA A 5' (Kpnl) (ig) (EcoRI) 20 and plasmid pKK310a opened with SphI and EcoRI (17).

The ligation mixture transforms E. coli strain JM109, isolates plasmid DNA, and ensures proper fusion by DNA sequencing. The plasmid with the correct sequence is named plasmid pKK400 (20).

Linker 19 encodes not only the remaining tendamate amino acid residues, but also the stem sequence portion that separates the sequence encoded by the tendamate and proinsulin gene and comprises a total of 30 residues from Table 2 corresponding to the 5 'end codons of the gene from the following 11 amino acid residues:

Thus, the Phe-Asn-AIa-Met-AIa-Thr-G1y-Asn-Ser-AIa-Arg 35 fusion protein in this stem sequence contains e.g.

amino acids methionine and arginine, which allow cleavage by halogen cyano or trypsin.

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An insert of about 1090 bp is isolated from plasmid pKK400 (20) by double digestion with SstI and Sphls and the DNA is ligated into plasmid pIJ702- (12) DNA opened with the same enzymes. Plasmid pGF1 (21) is obtained. Transform-5 is ligated with S. lividans strain TK 24 and plasmid DNA is isolated from thiostrepton-resistant transformants showing tendamate activity. All positive clones contain a 1090 bp SstI-SphI fragment from pGF1.

The construction of plasmid pGF1 (21) is shown in Figure 4.

Tendamate activity is determined by means of a plating test described both in Example 3 of EP-A1 0 161 629 and in DE-A-3 536 182, Example 15.

The fusion proteins encoded by plasmid pGF1 can be expressed in the usual manner. If the transformed S. lividans-TK 24 strain is incubated for 4 days at 28 ° C in shake flasks and the mycelium is separated from the culture medium by 20 centrifugation, the fusion protein can be detected in the clear supernatant as follows:

To 10-100 ul of culture supernatant add 20-200 ul of 15% trichloroacetic acid, enrich the precipitated protein by centrifugation, wash it and dissolve in assay buffer containing SDS (U. Laemmli, Natu re 227 (1970) 680). - 685). After incubation for two minutes at 90 ° C, electrophoretic separation is performed on a 10-17% SDS-polyacrylamide gel. A protein with a molecular weight of 19 kD is obtained, i.e. in the expected molecular weight range for the fusion protein of tendamate and proinsulin. The resulting fusion protein also reacts with both tendamate and insulin antibodies.

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table 1

Tendamate DNA sequence (code strand) and amino acid sequence

_ 5'-GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG

5 NH -Asp Thr Thr Vai Ser Glu Pro Ala Pro Ser Cys Vai 2

ACG CTC TAC CAG AGC TGG CGC TAC TCA CAG GCC GAC

Thr Leu Tyr Gin Ser Trp Arg Tyr Ser Gin Ala Asp 10 ts * «js

AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC

Asn Gly Cys Ala Glu Thr Vai Thr Vai Lys Vai Vai

TAC GAG GAC GAC ACC GAA GGC CTG TGC TAC GCC GTC

^ Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala Vai

GCA CCG GGC CAG ATC ACC ACC GTC GGC GAC GGC TAC

Ala Pro Gly Gin Ile Thr Thr Vai Gly Asp Gly Tyr

ATC GGC TCG CAC GGC CAC CCG CGC TAC CTG GCT CGC

Ile Gly Ser His Gly His Ala Arg Tyr Leu Ala Arg TGC CTT TAG-3 '

Cys Leu Stop codon 14 97549

Table 2

5 'AAT TCT GCA AGA 3' GA CGT TCT

(Asn) Ser Ala Arg 5 (EcoRI) B 1

TTT GTG AAC CAG CAC CTG TGC GGC TCC CAC CTA GTG GAA GCT CTC

AAA CAC TTG GTC GTG GAC ACG CCG AGG GTG GAT CAC CTT CGA GAG

Fhe Vai Asn Gin His Leu Cys Gly Ser Hls Leu Vai Glu Ala Leu 10

TAC CTG GTG TGC GGG GAG CGA GGC TTC TTC TAC ACA CCC AAG ACC

ATG GAC CAC ACG CCC CTC GCT CCG AAG AAG ATG TGT GGG TTC TGG

Tyr Leu Vai Cys Gly Glu Arg Gly Fhe Fhe Tyr Thr Fro Lys Thr C 1

15 CGC CGG GAG GCA GAG GAC CCT CAG GTG GGG CAG GTG GAG CTG GGC

GCG GCC CTC CGT CTC CTG GGA GTC CAC CCC GTC CAC CTC GAC CCG

Arg Arg Glu Ala Glu Asp Fro Gin Vai Gly Gin Vai Glu Leu Gly

GGG GGC CCT GGC GCA GGC AGC CTG CAG CCC TTG GCG CT G GAG GGG

CCC CCG GGA CCG CGT CCG TCG GAC GTC GGG AAC CGC GAC CTC CCC

20 Gly Gly Fro Gly Ala Gly Ser Leu Gin Fro Leu Ala Leu Glu Gly A 1

TCC CTG CAG AAG CGC GGC ATC GTG GAG CAG TGC TGC ACC AGC ATC

AGG GAC GTC TTC GCG CCG TAG CAC CTC GTC ACG ACG TGG TCG TAG

Ser Leu Gin Lys Arg Gly Ile Vai Glu Gin Cys Cys Thr Ser Ile 25

TGC TCC CTC TAC CAG CTG GAG AAC TAC TGC AAC TAA TAG TCG ACC

ACG AGG GAG ATG GTC GAC CTC TTG ATG ACG TTG ATT ATC AGC TGG

Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn

Sali 30 TGC AGC CA 3 'ACG TCG GTT CGA 5'

FstI (HindIII)

B1, C1 and A1 indicate the origin of the B, C and A chains of insulin monkey insulin.

Tue

Claims (8)

97549 A method for producing fusion proteins, characterized in that the structural gene of the desired protein is ligated to the 3 'end of a coding strand of an optionally modified tendonate gene, which gene encodes the tendonate shown in Table 1 or a modification thereof further capable of reversibly reacting with specific receptors. this gee-10 nirructure in Streptomyces host cells and isolating the secreted fusion protein from the culture supernatant. A method according to claim 1, characterized in that the tendamate gene is truncated from the 3 'end of the coding strand. 3. Gene construct, characterized in that the structural gene of another protein is linked to the 3 'end of the strand encoding the possibly modified tendonat gene, which gene encodes the tendonate shown in Table 1 or a modification thereof further capable of reversibly reacting with specific receptors. Gene construct according to Claim 3, characterized in that the tendamate gene is truncated at the 3 'end of the coding strand. Vector, characterized in that it contains a gene construct according to Claim 3 or 4. Streptomyces cell, characterized in that it contains the vector according to claim 5. 7. A fusion protein, characterized in that it contains an N-terminal portion of the tendamate sequence shown in Table I or a modification thereof which is further capable of reacting reversibly with specific receptors. Use of a fusion protein according to Claim 7 or obtainable according to Claim 1 or 2, characterized in that it is carried out for the preparation of an optionally modified tendamate and / or a fusion partner thereof. 97549