SK110191A3 - Method of production of foreign proteins in streptomycets - 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

Method of production of foreign proteins in streptomyces

Technical field

It is known from European patent application publication number (EP-A) 0 289 936 that fusion proteins are produced by coupling the structural gene for the desired protein to the 3'-end of the coding strand or the modified tendamistate gene, which gene structure is expressing in the host cell streptomyces and secreting the fusion protein is isolated from the medium. In a preferred embodiment, the tendamistate gene is truncated at the 3'-end. Truncation sites for restriction enzymes BstEII in the region of triplets 31 and 32, StuI in the region of triplets 43 and 44, and Sau3A in the region of triplets 52 and 53 are used for truncation.

In a further development of this inventive idea, it has already been proposed to produce a fusion protein in which the proportion of tendamistate is followed by a truncated proinsulin whose C-chain consists of only one or two lysine residues (miniproinsulin). As a further development, it has been proposed that the proportion of tendamistate in such fusion proteins be shortened (European application EP-A 0 367 163 published May 9, 1990).

Surprisingly, it has now been found that fusion proteins with a very short proportion of tendamistate are stable in the streptomyces cell and wash out! to the media. The fusion proteins thus obtained behave as mature proteins due to the very short chain of tendamistat and exist generally in the medium in the correct tertiary structure.

EP-A 0 177 827 discloses a synthetic signal sequence for transferring a protein to an expression system, characterized in that the DNA corresponds to a substantially natural signal sequence, but has one or more endonuclease step sites that are not contained in the natural DNA. When the gene for the protein to be transferred is coupled to such a DNA sequence, the fusion gene is incorporated into the vector and thereby transforms a host cell that transports the expressed protein from the cytoplasm, prokaryotic or viral proteins can be produced in prokaryotic and eukaryotic cells. As an example of a periplasmic protein, alkaline phosphatates show that when expressed in E. coli it is preferred that, following the pre-sequence, codons for about the first 40 amino acids of alkaline phosphatase be inserted before the structural gene for the desired protein. In many cases, however, fewer additional amino acids, for example about 10, preferably about 5, will suffice. The corresponding monkey proiusulin fusion protein has been transported about 90% into the periplasmic space.

It has also been proposed (WO 91/03 550 of 21 March 1991) to produce fusion proteins by constructing a mixed oligonucleotide that encodes a ballast portion of the fusion protein, which oligonucleotide is introduced into the vector so that it is operably coupled to a regulatory region and the structural gene for the protein of interest is transformed with the plasmid population thus obtained. suitable host cells and those clones that have a high yield of fusion protein are selected.

The oligonucleotide preferably consists of 4 to 12, in particular 4 to 8, triplets.

It has also been attempted to produce fusion proteins with a short ballast fraction. For example, a gene fusion that encodes a fusion protein of the first 10 amino acids of β-galactosidase and somatostatin has been produced. However, it has been shown that this short β-galactosidase fragment is not sufficient to protect the fusion protein from self-host protease degradation (US 4,366,246, 15, paragraph 2). Accordingly, EP-A 0 290 005 and 0 292 763 disclose fusion proteins whose ballast portion consists of a β-galactosidase fragment of more than 250 amino acids.

SUMMARY OF THE INVENTION

Unexpectedly, it has now been found that fusion proteins, consisting of about the first 10 amino-terminal amino acids of tendamistate and the desired protein, for example proinsulin, are stable in streptomyces host cells and are secreted into medium from which they can be obtained in high yields. It is surprising that this also applies to relatively small proteins such as mini-proinsulins.

About 10 amino acids means that less amino acids are possible, for example the first 7 N-terminal amino acids of tendamistate, but preferably no more than

10. Preferred are fusion proteins in which the proportion of tendamistate is at the 7 and / or 9 position of proline (as in the natural sequence).

It is, of course, also possible, in accordance with known or proposed embodiments, to select a larger ballast portion of tendamistate, but naturally the advantage of a small ballast is more and more lost.

It is possible and even advantageous for the natural amino acid sequence of the tendamistat moiety to vary, that is, when the amino acids are exchanged, deleted or not introduced as they occur in the natural amino acid sequence. It is further possible to vary the amino acid sequence of the signal peptide.

Especially preferred fusion structures can be readily ascertained based on the inventive idea by simple preliminary experiments.

Furthermore, it is also possible to practice the invention in other gram-positive bacterial cells, for example in bacillus or staphylococcal cells, using signal sequences recognized by these hosts.

The fusion proteins obtained according to the invention are present. in a medium in dissolved form, which provides a number of advantages for further processing and purification. Thus, for example, further enzymatic treatment can be carried out to cleave the ballast portion immediately with the secretion product, without the need for processing procedures which are necessary for the insoluble fusion proteins. It is also possible to first concentrate or purify, for example by means of affinity chromatography, but also ultrafiltration, precipitation, ion exchange chromatography, adsorption chromatography, gel filtration or high pressure liquid chromatography before further processing.

DETAILED DESCRIPTION OF THE INVENTION

The invention is explained in more detail in the following examples.

The starting material for the plasmid constructs is the plasmid pKK500, which was proposed in EP-A 0 367 163. This plasmid differs from the plasmid pKK400, known from EP-A 0 289 936, by replacing the proinsulin gene with an analogue gene which encodes only the amino acid instead of the carbon chain. lysine, as well as the introduction of a terminator sequence joining this mini-proinsulin gene. Tables 1 and 2 show the mini-proinsulin gene and the terminator sequence.

Table 1

B ¹ 10

ASN SER ASN GLI PRESS PHE VAL ASN GLN HIS LEU CIS GLI 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 TIR LEU VAL CIS GLI GLU ARG GLI 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 C A ¹ 40 TIR THR PRO LYS THR LYS GLY ILE VAL 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 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 GGA CTC TTG ATG ACG TTG ATC ATT

GTC GAC CTG CAG CCA CAG CTG GAC GTC GGT TCG

Sali (Hindu)

T a b at 1 k and 2

5 'OGATTAAACOGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAGATTTTCAACGTGGA

GCTATTTGGCTATGTTAATTTCOGAGAAAACCTCGAAAAAAAAACCTCTAAAAGTTGCACCTAG-5 ¹

The plasmids pKK400 and pKK500 contain! in the signal sequence of the α-amylase inhibitor gene, the steppe site XmalII (in the region of the triplet -5 to -7).

Example 1

Plasmid pKK500 is opened with the restriction enzymes EcoRI and XmalII and the large fragment is separated on a 0.8% agarose gel by gel electrophoresis and isolated by electroelution. This fragment was ligated with a DNA fragment (1) (SEQ ID NO: 1) synthesized by the phosphoramidite method

-5 -1

Ala Gly for Ala Ser Ala 5 'G GCG GGG CCG GCC TCC GCC 3 ' CCC GGC CGG AGG CGG

(XmalII)

Asp Thr Thr wall Ser Glu for GAC ACG ACC GTC TCC GAG CCG 3 GTG TGC TGG CAG AGG CTC GGC TTA 5

(EcoRI) and the ligation mixture is transformed into E. coli. Plasmid pKK510 is obtained. This encodes a preproinsulin in which the tendamistate signal sequence is followed by the first 7 amino acids of the tendamistate and then the mini-proinsulin chain.

Example 2

Analogously to the method for converting plasmid pHH400 to the expression plasmid pGF1 described in EP-A 0 289 936, the plasmid pKK510 is converted into the expression plasmid pKF1:

The isolated DNA of plasmid pKK510 was digested with the restriction enzymes SpHI and SstI, and a small fragment with the fusion gene was isolated. The commercial expression plasmid pIJ 702 (obtainable from the John Innes Foundation, Norwich, England) is digested with the same enzymes and a large fragment is isolated. The two isolated fragments were ligated, the ligation mixture transformed in S. lividans TK24, and a plasmid was isolated from thiostrepton-resistant white transformants (i.e., unable to form melanin). Clones carrying the inserted information are tested in shake culture for the ability to form fusion proteins.

Expression of the encoded fusion protein is carried out in a manner known per se: the transformed strain is incubated for 4 days at a temperature in excess of 25 ° C in a shake flask and the mycelium is separated by centrifugation from the culture solution. The formed fusion protein can be visualized in the culture medium after electrophoresis of 20 μm of culture filtrate in a 15% polyacrylamide gel by staining with CO COOMASSIE-blue as an additional band of protein that did not appear in the control experiment to which the strain was only transformed with pIJ 702.

When the culture filtrate is treated with lysylendoproteinase, it can be detected by Des- (B30) -Thr-insulin gel electrophoresis, which is verified by authentic control.

Further, the fusion protein in the culture filtrate can be detected with either insulin antibodies in the Immuno-Blot or insulin-RIA assay.

Example 3

The procedure was as in Examples 1 and 2, but using the synthetic fragment (2) with SEQ ID NO: 2.

-5 -1

Ala Gly for Ala Ser Ala 5'G CCG GGG CCG GCC TCC GCC 3 ' CCC GGC CGG AGG CGG

/ XmalII /

Asp Thr Thr wall 5 Ser Glu for Asp for GAC ACG ACC GTC TCC GAG CCC GAC CCG 3 ' CTG TGC TGG CAG AGG CTC GGG CTG GGC TTA 5 '

(EcoRI) to obtain plasmids pKK320 and pKF2, respectively.

These plasmids encode a fusion protein that differs from the protein of Examples 1 and 2 in that the first 7 amino acids of the tendamistate are followed by asparagine (instead of the natural amino acid alanine) and then the ninth amino acid in the tendamistate, proline. Thus, by exchanging alanine for asparagine, an additional positive charge is introduced into the ballast portion of the fusion protein. Surprisingly, yields of about 20 to 30% higher than in Example 2 are obtained.

Example 4

The procedure of Examples 1 and 2 is followed, but the synthetic fragment (3) of SEQ ID NO: 3 is used

-5 -1

Ala Gly for Ala Ser Ala 5 'G GCC GGG CCG GCC TCC GCC 3 ' CCC GGG CGG AGG CGG

/ XmalII /

Asp Thr Thr wall Ser Glu for Ala for GAC ACG ACC GTC TCC GAG CCC GCA CCG 3 ' CTG TGC TGG CAG AGG CTC GGG CGT GGC TTA 5 '

whereby plasmids pKK330 and pKF3, respectively, are obtained. These plasmids differ from the plasmids of Examples 1 and 2 in that they encode the first 9 natural amino acids of tendamistate. Compared to Example 2, yields of about 10% higher are obtained.

Example 5

The fusion protein, encoded by pKK500, contains a linker sequence encoding the amino acids Asn-Ser.Asn-Gly-Lys between the tendamistate fraction and the proinsulin B-chain. The replacement of this terminal lysine and lysine, representing the carbon chain, with arginine is carried out as follows. The singular cleavage site Styl in the region of codon B30 to A1 of the proinsulin sequence is used.

The isolated plasmid DNA, derived from pKK500, was digested with Styl, digested to remove the overlapping ends of the SI nuclease, and the excess nuclease was extracted with phenolic chloroform. The linearized plasmid is then further digested with EcoRI, the large fragment separated by electrophoresis and isolated by electroelution. This fragment is ligated with the synthetic fragment (4) of SEQ ID NO: 4 /

BI 10

same time Ser same time Gly Arg Phe wall same time A boat His Leu Cys Gly Ser His AAT TCG AAC GGC CGC TTC GTC AAC CAG GAC CTG TGC GGC TCG CAC GC TTG CCG GCG AAG CAG TTG GTC GTG GAC ACG GGG AGC GTG

/ EcoRI /

- 11 20 30

Leu wall Glu Ala Leu Tyr Leu wall Cys Gly Glu Arg Gly Phe Phe CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC CTC TTC GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC CGC CCG AAG AAG B / 3 C i C / B31 /

Tyr Thr for Lys Thr Arg TAC ACC CCC AAG ACC CGC ATG TGG GGG TTC TGG GGG

and the ligation mixture is transfected in E. coli. The desired clones were screened by restriction analysis of the plasmid obtained, using the new SstIII cleavage site formed. Next, the entire SphI-SstI fragment is sequenced.

For the expression of the encoded fusion protein, the fragment examined by sequence analysis was ligated in the pIJ 702 vector digested with the same enzymes to produce the expression vector pGF4.

Demonstration of the fusion protein encoded by pGF4 and secreted can be carried out by both a plate assay using an α-amylase inhibitor (EP-A 0 161 629), Example 3, and a shake culture medium analogously to Example 2.

Example 6

If, as in Example 5, the fragment (4) is introduced into vector pKK510, 520, 530, vectors pKK610, 620 and 630 are obtained. Incorporation of such SphI-SstI fragments with the sequence encoded for the fusion proteins into pIJ 702 provides pKF11 expression vectors. , 12 and 13.

The expression of secreted fusion proteins was tested according to Example 2.

Example 7

To increase the expression of the plasmid derivatives pIJ 702, the melamine promoter is removed from this digestion with PstI and SphI and replaced with the synthetic fragment [5] (SEQ ID NO: 5).

PstI Were

20 30 40

CTGCAGTGATCAGGGGGAGCCTTGTGCGAATTTCČGTTACGGGTTTGGGTGGTAGGG GACGTCACTAGTCCCCCTGGGAACACGCTTAAAGGCAATGCCCAAACCCACCATCCC

SphI

70 80

ACGCACCCGAAGAGGAGGCCCCAGCATGC TGCGTGGGCTTCTCCTCCGGGGTCGTACG

This creates a tandem construct from a synthetic promoter and a tendamistate promoter. The plasmid is designated pGRUO.

When synthetic fragments (1), (2), and (3) are used in pGRUO after digestion with SphI and SstI, the expression vectors pGR200, 210, and 220 result in analogy. 260 and 270.

Example 8

If human insulin is to be made from the preinsulins by combining trypsin or the same carboxypeptidase B enzyme, it is preferable to cleave the amino-terminal ballast portion rapidly during the stepping reaction in order to promote it towards B31 (Arg) -insulin. To this end, it is possible to modify amino acids before amino acid B1 (Phe):

The procedure of Example 1 is followed and the plasmid pKK500 is opened with the restriction enzymes EcoRI and DraII. The original fragment is then replaced with a DNA fragment (6) synthesized by the phosphoramidite method (SEQ ID NO: 6).

BI

same time Ser Ala Arg Phe wall same time 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 / DralII /

Cloning in E. coli and expression in Strptomyces lividans was carried out according to Example 1 and Example 2 respectively, respectively, resulting in the plasmid pKK640 and the expression plasmid pKF14, respectively.

The plasmid obtained according to Example 5 (after the built-in fragment (4)) can be used analogously. Plasmid pKK650 and pKF15, respectively, are obtained.

Claims (8)

Method for producing fusion proteins, characterized in that the structural gene for the desired protein is coupled to the codons for the signal sequences and about the first ten amino-terminal amino acids of the tendamistate, the gene structure is expressed in a streptomyces host cell for expression and secreted the fusion protein is isolated from the medium, wherein the tendamistate gene sequences can be modified. 2. A gene structure comprising a signal sequence and about the first ten codons for the tendamistate and a structural gene for - another protein. * 3. The gene structure of claim 2, wherein the tendamistate portion removes, exchanges or adds codons for amino acids. 4. The gene structure according to claim 2 or 3, characterized in that a linker sequence is arranged between the tenda state gene and the structural gene for the protein of interest. A vector comprising the gene structure of claim 2, 3 or 4. A streptomyces cell comprising the vector of claim 5. 7. A fusion protein having an N-terminal portion of about the first ten amino acids of tendamistate coupled or, if appropriate, a pre-must sequence to the desired protein. Use of the fusion protein of claim 7, or the fusion protein obtainable of claim 1, for producing the desired protein.