LT3686B - Process for preparing fused proteins, gene structure, process for transformation of hybride and streptomycetes cells - 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

BACKGROUND OF THE INVENTION The invention relates to the field of biotechnology and relates to a method for obtaining fusion proteins, a gene structure and a hybrid, and a method for transforming streptomycetes cells.

A known method of producing fusion proteins is to attach a structural gene of the desired protein to the 3'-end of the coding strand, expressing this gene structure into streptomycete hosts and isolating the secreted fusion protein from supernatant fluid (EP-A089936). . In a preferred embodiment, the tandem gene is truncated at the 3'-terminus. Truncation sites for the restriction enzymes BstEII triplets 31 and 32, StuI triplets 43 and 44, and Sau3 A triplets 52 and 53 are used for truncation.

To further elaborate on this idea, it has been proposed to obtain a fusion protein with a truncated proinsulin whose C-chain is composed of only one or two lysine residues ("mini-proinsulin"). It was further proposed to shorten the tandem portion in similar fused proteins (EP-A 367163).

It has recently been unexpectedly found that fusion proteins with a very short tandem moiety in the streptomycete cell are stable and released into the medium. The resulting fusion proteins, due to their very short tandem chain, behave like "mature" proteins and exist in the medium in the form of a regular tertiary structure.

Synthetic signaling DNA sequence for the transport of proteins to expression systems is known from EP-A-0177827, which differs in that this DNA essentially corresponds to the natural signaling sequence but has one or more endonuclease cleavage sites that are not present in the natural DNA. If the gene for the transported protein is attached to such a DNA sequence, this fused gene is inserted into a vector and thus transformed into a host cell that transports the released protein from the cytoplasm, thereby obtaining eukaryotic, prokaryotic, or viral proteins in prokaryotic and eukaryotic cells. An example of a periplasmic protein, alkaline phosphatase, shows that E.coli expression is preferably introduced after about 40 of the first amino acid codon sequence of the alkaline phosphatase before the structural gene of the desired protein. However, in most cases a smaller number of additional amino acids, e.g. The corresponding bound protein with monkey proinsulin is transported up to 90% into the periplasmic space.

A method for producing fusion proteins by constructing a hybrid oligonucleotide encoding the ballast portion of a fusion protein (PCT No. 91/03550) has been proposed. This oligonucleotide is introduced into a vector such that it is functionally linked to the regulatory domain and the structural gene of the desired protein; the resulting plasmid population is transformed into suitable host cells and selected clones with high yields of encoded fusion protein. In this case, the oligonucleotide consists mainly of 4-12 triplets, in particular cases 4-8 triplets.

Attempts have been made to obtain fused proteins with short ballast. For example, a gene fusion that encodes a fusion protein from the first 10 amino acids of β-galactosidase and somatostatin was obtained. However, this short β-galactosidase fragment did not appear to be sufficient to protect the spliced protein from host proteases (US-A 4306246, column 15, paragraph 2). EP-A-0290005 and EP-A-0292763 disclose a fusion protein having a β-galactosidase moiety of 250 amino acids in a ballast.

Most recently, fusion proteins consisting of tandem's first approximately 10 N-terminal amino acids and a desired protein, such as proinsulin, have unexpectedly appeared to be stable in streptomycete host cells and capable of being released into the medium from which they can be isolated in high yield. This is important for relatively small proteins such as "mini-proinsulins".

&Quot; Approximately 10 amino acids " means a lower number of amino acids, such as the first N-terminal amino acids of the tandem 7, but most preferably not more than 10. Preferably, when the tandem portion of the fusion protein is in position 7 and / or 9je ( as in the natural sequence) is proline.

Of course, the known and proposed variants (EP-A-0367163) allow for the choice of a large tandem ballast, while of course reducing the advantage of a small "ballast".

It is possible, and even advisable, to alter the natural sequence of the tandem moiety at position 7-10, i.e. substituted with non-naturally occurring amino acids. In addition, the amino acid sequence in the signal peptide can be varied.

The essence of the invention is that, by means of simple preliminary tests, particularly suitable joint structures can be easily identified.

Further, the inventive ideas can be implemented in other Gram-positive bacterial cells, such as bacilli or staphylococcal cells, using signal sequences that are "recognized" by these hosts.

The fusion proteins obtained according to the present invention are dissolved in the medium, which is very convenient for further processing and purification. For example, it is possible to carry out the enzymatic treatment directly with the aid of a secretory product, separating the ballast and avoiding the processing steps required for insoluble fused proteins. Prior to further treatment, condensation or purification can be carried out, for example, by affinity chromatography as well as by ultrafiltration, precipitation, ion exchange, adsorption chromatography, gel filtration or high pressure liquid chromatography.

The following examples further illustrate the invention.

In the construction of the plasmid, the starting material was the plasmid pKK500 proposed in EP-A 0367163. This plasmid differs from the known plasmid pKK400 of EP-A-0289936 in that the proinsulin gene is replaced by an analogous gene which encodes only the amino acid li '. .ina, and also because a terminator is introduced after this "mini-proinsulin" gene. As an appendix to the description, Tables 1 and 2 of EP-A 0367163 depicting the mini-proinsulin gene, respectively, are provided.

Plasmids pKK400 and pKK500 contain an XmaIII cleavage site (5-7 region of the triplet) in the signaling sequence of the α-amylase gene.

example

Plasmid pKK500 cleaves a large fragment by restriction enzymes EcoRI and XmaIII and gel electrophoresis on 0.8% agarose gel, which is then separated by electroelution. This fragment is ligated with the DNA fragment (1) synthesized by phosphoramidate (SEQ ID NO: 1) and transformed into E.coli.

-5 -1

Ala Gly Pro Ala Ser Ala 5 'C, GCC GGG GGC GCC TCC GCC 3' CCC GGC CGG AGG CGG (XmaIII)

Asp Thr Thr Or Ser Glu Pro

GAC ACG ACC GTC TCC GAG CCG 3 '

CTG TGC TGG CAG AGG CTC GGCTTA A 5 '(EeoRI)

The plasmid pKK510 is obtained. It encodes a pre-proinsulin which is followed by a tandem signaling sequence followed by the first 7 amino acids followed by a mini-proinsulin chain.

example

In an analogous manner to the method described in EP-A 0289936 for converting plasmid pKK400 into pGF1 expression plasmid, pKK510 is converted to pKF1 expression plasmid:

isolated DNA-plasmid pKK510 was cleaved by restriction enzymes Sphl and SstI and isolated a small fragment with the fused gene. The commercially available expression plasmid pIJ 702 (John Innes Foundation, Norwich, England) was cleaved with the same enzymes and isolated a large fragment. These two isolated fragments are ligated, the ligated mixture is transformed into 5. lividans TK24, and a plasmid is isolated from thiostrepton-resistant, white (i.e., inability to produce melanin) transformants. The clones, which have an inserted insert, are cultured in shake culture and assayed for fusion proteins.

Expression of the encoded fusion protein occurs in a known manner. Incubation of the transformed strain for 4 days in shaker flasks at temperatures above 25 ° C and separation of the micelle from the culture fluid by centrifugation results in COOMASSIE blue staining to provide an additional protein band that does not appear in the control experiment with strain transformed with pIJ 702 alone. can be detected after electrophoresis in 20 µm supernatant fluid and culture fluid filtrate on 15% polyacrylamide gel.

If the culture fluid filtrate is exposed to lysylendoproteinase, I) es- (B30) -Thr-insulin can be detected by gel electrophoresis, which is confirmed by authentic controls.

Further, the fusion protein can be detected by the use of insulin antibodies either by immunoblocking or by radioimmunoassay of the insulin in the filtrate of the culture fluid.

example

According to the methodologies described in Examples 1 and 2, using only synthetic fragment (2) with SEO II) N (): 2

-5 -1

Ala Gly Pro Ala Ser Ala

5 'G GCC GGG ccg gcc tcc gcc 3' CCC GGC CGG AGG CGG (XmaIH)

Asp Okr Thr Or Ser Glu Pro Asp Pro G AG ACG ACC GTC TCC GAG CCC GAC CCG 3 '

CFG IX i C TGG CAG AGG CTC GGG CTG GGC TTA A 5 '(EcoRI) receives plasmids pKK320 and pKF2, respectively.

These plasmids encode a fusion protein that differs from that described in Examples 1 and 2 in that the first 7 tandem amino acids are followed by asparagine (instead of the natural amino acid, alanine), and the ninth amino acid in tandem is proline. Thus, the replacement of alanine with asparagine introduces an additional positive charge into the ballast portion of the bound protein. Unexpectedly, 20-30% more of the desired product is available than in Example 2.

example

According to the procedures described in Examples 1 and 2, but using the synthetic fragment (3) with SEO II) N (): 3

-5 -1

Ala Gly Pro Ala Ser Ala

5 'G GCC GGG CCG GCC TCC GCC 3 CCC GGC CGG AGG CGG (XmaIII)

Asp Thr Thr Va] Ser Glu Pro Ala Pro

GAC ACC. ACC GTC TCC GAG CCC GCA CCG 3 '

C1C ICC TCC CAC ACC CTCi CCG CCT CCC TTA A 5 'receives plasmids pKK320 and pKF3, respectively. These plasmids differ from the plasmids obtained in Examples 1 and 2 in that they encode the first 9 natural amino acids in tandem. Compared to Example 2, the yield is nearly 10% higher.

example

The fusion protein encoded by pKK500 has a linker sequence between the tandem moiety and the proinsulin Bgrand that encodes the Asn-Ser-Asn-Gly-Lys amino acids. The rear Lys and the forming C-chain are replaced by Lys as described below. For this purpose, the single insertion site Styl is used in the proinsulin sequence at codon B30 to Al.

The isolated DNA-plasmid pKK500 is cleaved with Styl by Styl to remove protruding ends, and the excess is extracted with a phenol-chloroform mixture. The resulting linear plasmid is again cleaved by EcoRI, electrophoresed to isolate the large fragment, and isolated by electroelution. This fragment is ligated to the synthetic fragment (4) (SEQ ID NO: 4)

JU 10

Asn Ser Asn Gly Arg Phe Vai Asn Cln His Leu Cys Cly Ser His ATI 'TCC. AAC CCC CCC TTC. CCT AAC CAG CAC. CTG TCC GGC TCC CAC

CC'ITCCCC GCC AAC CAC TTG GTC CTG GAC ACG CCG AGC GTC (EcoRI)

30th

Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe ere C'l'G CAC. CCC CTC TAC CTG CTG TCC CCC CAC CCC GGC TTC TTC

CAC CAC ere CCC CAC ATC CAC CAC ACG CCC CTC GCG CCG AAC AAC

B30 C (B31)

Tyr Thr Pro Lys Thr Arg

TAC ACC CCC AAC ACC CCC

ATG TGG GGG I I C TCC CCC and after ligation the mixture is transformed into E.coli. Preferred clones are screened by restriction plasmid analysis of the available plasmid and utilized the newly formed SstlI cleavage site. The entire SphI-Sstl fragment is sequenced.

For expression of the encoded fusion protein, the fragment is ligated into the vector pIJ 702. digested with the same enzymes to yield the expression vector pGF4.

The encoded pGF4 and secreted fusion protein can be detected, first, by assaying on plates with an α-amylase inhibitor (EP-A 0161629, Example 3) and second, analogously to Example 2, from cultured supernatant fluid by continuous shaking.

example

When the fragment (4) is introduced into the vectors pKK51 (), 520 and 530 according to the procedure described in Example 5, the resulting vectors pKK610, 620 and 630 are obtained. The SphI-SstI fragments, respectively, inserted into the pIJ 702 , 12 and 13. The expression of secreted fused proteins is verified according to Example 2.

example

The plasmid pIJ 702 is amplified by expressing the melanin promoter with PstI and Sphl and replacing it with fragment (5) (SEQ ID NO: 5)

PstI Bell

K) 20 30 40 50

CTG (: a <'.TGATt: ac k k; gg acccttgtgcc; aatttgcgttacgggtttgggtggtaggg GACGTCATCAC iTCCCCCTGGGAACACGCTTAAAGGCAATGCCCAAACCCACCATCCC

Sphl

70 80

ACG (? AG (X X i AAC AC iG A (iGGCCCAGCATGC TGC X i'IT i ('. C'. (TT ('ΊΧ X YCC GGGGTCGTACG

This produces a tandem construct from both synthetic and tandem promoters. The plasmid is named pGRl 10.

By inserting synthetic fragments (1), (2) and (3) into pGR.110 after cleavage with SphI and SstI, expression vectors pGR200, 210 and 220 are obtained. Similarly, expression vectors pGR250, 260 and 270 are obtained with the aid of fragment (4). .

example

If human insulin is to be obtained from insulin precursors by a combination of trypsin or a similar enzyme and carboxypeptidase B, it is preferable to rapidly separate the ballast with the amino terminus during the separation reaction to induce degradation reactions in the direction of B31 (Arg) -insulin. For this purpose, a modification of the pre-amino acid JV (Phe) is proposed.

The plasmid pKK500 was cleaved by the restriction enzymes EeoRI and DralII according to the procedure described in Example 1. The parent fragment is then replaced by the phosphoramidite method into the synthetic DNA fragment (6) (SEQ ID NO: 6)

JV

Asn Ser Ala Arg Phe Or Asn Gln His Leu Cys Gly Ser His Leu 5 'ΑΛΊ' TGG (K '(' ('GG TTC GTC AAG CAG CAC CGT TGC GGC TCG CAC CTC 3'

3 'GG (X> G (' iGG AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG 5 '(EeoRI) (DralII)

Cloning in E.coli cells and expression in Streptomyccs lividcins cells are performed according to Examples 1 and 2, respectively. The plasmids pKK640 and pKF14 are obtained respectively.

Similarly, the plasmid obtained in Example 5 (inserting fragment (4)) can be treated to give plasmids pKK650 and pKF15, respectively.

APPENDIX (From EP-A 0367163) Table

JV

K)

Asn Ser Asn AATTCG AAC GC TTG (EcoRI) Gly GGC ('(' G Lys AAG TTC Phe TTC AAG Or Asn GTC AAC CAG TTG Gln CAG GTC His CAC GTG Leu CTG GAC Cys TGC ACG Gly GGC CCG Ser TCG AGC His CAC GTG 20th 30th Leu Vai Glu Ala Eeu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe CTC GTG ΟΛΟ GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC G AG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG C Al 40 Tvr Thr Pro Lys Thr Lys Gly Ile Or Glu Gln Cys cys Thr Ser TAC ACC CCC AAG ACC AAG GGC ATC GTG GAG CAG TGC TGT ACG TCC ATG TGG GGC i ttc TGG TTC CCG TAG CAC CTC GTC ACG ACA TGC AGG

Ile Cys Ser Eeu 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 GTC CAG ACG AGG GAC ere CFG GAC GAG ATG GTC ' CAG CCA CTG GGT TCG GAG A CTC TTG ATG ACG TTG ATC ATT

(HindlII)

Hall table

5'-C (i ATAAACCC i ATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAG A (iCl 'ΛΤΓΓί ϊ (iCTATGTTAATTTCCGAGGAAAACCTCGGAAAAAAAAACCTCT

TTTTCAACGTY i {i ATC

AAAAC ί ΊΊΧICACCTAG-5 '

SEO II) NO: 1 SEQUENCE TYPE:

SEQUENCE LENGTH: CHAIN SHAPE: TOPOLOGY: FRAGMENT TYPE:

nucleotide with corresponding protein base double strand with conserved recognition linear inner fragment

ORIGIN synthetic DNA

G GGG GGG CGG GCC TCC GCC GAC ACG ACC GTC TCC GAG CCG

GGC 'GGG GGG AGG CGG CTG TGC TGG CAG AGG CTC GGC TTA A

Ala Gly Pro Ala Ser Ala Asp Thr Thr Or Ser Glu Pro Asn

SEO II) N (): 2 SEQUENCE TYPE:

SEQUENCE LENGTH: CHAIN SHAPE TOPOLOGY: FRAGMENT TYPE: ORIGIN:

nucleotide with corresponding protein double strand of bases with a conserved recognition sequence linear inner fragment synthetic DNA

G GGG

GGG CCG GCC TCC GCC GAC ACG ACC GTC TCC GAG CCC GAC GGG GGG CGG AGG CGG CTG TGC TGG CAG AGG CTC GGG CTG

Ala {ily Pro Ala -5

CCG TTTA

GGG

Ser Ala Asp Thr Thr Or Ser Glu Pro Asp 1 5

Pro Asn 10

SEO ID NO: 3 SEQUENCE TYPE:

SEQUENCE LENGTH: CHAIN SHAPE: TOPOLOCATION: FRAGMENT TYPE: ORIGIN:

nucleotide with corresponding protein double strand of bases with a conserved recognition sequence linear inner fragment synthetic DNA

C GGG GGG GGG GCC 'TGC GCC GAC ACG ACC GTC TCC GAG CCC GCA CTG GGC 'CGG AGG CGG CTG TGC TGG CAG AGG CTC GGG CGT Ala (ily Pro Ala Ser Ala Asp Thr Thr Or Ser Glu Pro Ala -5 1 5 GGG- () ('. (' T 1Ά A

Pro Asn

SEQ ID NO: 4

SEQUENCE TYPE: Nucleotide with corresponding protein

SEQUENCE II. (IIS: 108 bases

CHAIN SHAPE: Dual chain with persistent recognition sequence

TOPOLOGY: Linear

FRAGMENT TYPE: Internal fragment

MEASURE II: Synthetic DNA

ATT TCG AAG GGC CGC TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC

Asn GC TIG Ser Asn CCG Gly GCG AAG CAG TTG GTC. GTG GAC ACG CCG AGC GTG Arg 5 Phe Or Asn Gln His 1C Leu 1 Cys Gly Ser His 15th CTC GTG GAC. GCC CTC TAC. CTG GTG TGC GGG GAG CGC GGC. TTC TTC G AG CAC CTC CGG GAG ATG GAC. CAC AGG CCC CTC GCG CCG AAG AAG Leu Or Glu Ala Leu Tyr Leu Or Cys Gly Glu Arg Gly Phe Phe

25 30

TAC ACC CCC AAG ACC CGC ATG 'L'GG GGG TTC TGG GCG Tyr I h r Pro lys Thr Arg 35

SEO ID NO: 5

SEQUENCE TYPE: Nucleotide with

SEQUENCE LENGTH: 86 bps

CHAIN SHAPE: TOPOLOGY: FRAGMENT TYPE: ORIGIN:

double strand linear inner fragment synthetic DNA

CTGCAGTGAT CAGGGGGACC CTTGTGCGAA TTTCCGTTAC GGGTTTGGGT 50 GGTAGGGAGG CACCCGAAGA GGAGGCCCCA GCATGC 86

SEO ID NO: 6

SEQUENCE TYPE: Nucleotide with corresponding protein

SEQUENCE II, GIS: 45 bases

CHAIN SHAPE: Dual chain with persistent recognition sequence

TOPOLOGY: Linear

FRAGMENT FOOT: Internal fragment

ORIGIN: Synthetic DNA

AAT TCG GCC CGC TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC CTC GC CGG GCG AAC! CAG TTG GTC GTG GAC. ACG CCG AGC GTG

Asn Ser Ala Arg Phe Vai Asn Gln His Leu Cys Gly Ser His Leu

Claims (6)

CLAIMS 1. A method for producing a fusion protein comprising binding a structural gene of the desired protein to a signal sequence codon and the first about ten amino acids of the tandem, expressing the gene into a host streptomycete for expression and secreting the secreted fusion protein from the supernatant fluid. sequences can be modified. 2. A method of obtaining an iene structure comprising binding the signal sequence and the first ten tandem codons to the structural gene of another protein. 3. The method of claim 2, wherein the tandem portion omits, replaces, or introduces amino acid codons. 4. The method of claim 2 or 3, wherein the linker sequence is introduced between the tandem gene and the structural protein of the desired protein. 5. A method of producing a hybrid comprising introducing the gene structure according to claim 2, 3 or 4 into a streptomycetic vector. 6. A method of transforming streptomycetes cells, comprising introducing the vector into these cells according to claim 5.