FR2599380A1 - Vector for exporting proteins in Escherichia coli, transformed bacteria and method for preparing proteins - Google Patents

Abstract

The invention relates to a DNA unit intended for exporting a specific protein in E. coli, characterised in that it comprises at least the following sequence: - PhoS corresponds to the PhoS gene or to a portion of this gene which is responsible for its export, the said gene being devoid of stop codon, - Sc corresponds to a sequence encoding one or more amino acids which are cleavable chemically or enzymatically, releasing the product of the X gene, - X corresponds to the gene encoding the specific protein and devoid of a coding sequence similar to the Sc sequence.

Description

 The present invention relates to a method for the export of proteins to Escherichia coli.

 It has been known for some time now to synthesize proteins with E. coli bacteria using expression vectors such as pBR322.

However, one of the disadvantages of the methods is that the proteins thus synthesized are not exported by the bacteria.

 This leads to difficulties in the purification of the synthesized protein.

This is why the present invention relates to a method for exporting proteins to
E. coli which makes it possible to purify the protein obtained very easily.

More particularly, the present invention relates to an export block of a protein determined in E. coli characterized in that it comprises at least the following sequence

<tb> Phos <SEP> Sc <SEP> X
<tb> - PhoS is the PhoS gene or part of that gene
ensuring its export, said gene being devoid of
stop codon, - Sc corresponds to a coding sequence for one or more
chemically or enzymatically cleavable amino acids
to release the product of the X gene, X corresponds to the gene coding for the determined protein
and lacking a coding sequence similar to the Sc sequence.

 The export block according to the invention will preferably be inserted into a plasmid vector of E. coli, which will include an origin of functional replication of this bacterium.

 Of course, the vectors according to the present invention may comprise other elements which are now known in the field of recombinant DNAs, namely in particular the different elements necessary for the expression of the PhoS and X genes, that is to say essentially a promoter, a translation initiation site, as well as possibly different sequences allowing regulation if necessary.

 In addition, it may be advantageous to provide a marker gene coding, for example, for resistance to an antibiotic such as the Ampr or Tetr genes.

 These various elements are known and will not be described in detail below.

 With regard to the essential elements characteristic of the present invention, the use of the PhoS gene has many advantages. Indeed, it is an affine protein for phosphate whose synthesis and structure have very interesting characteristics.

 Firstly, the production of this protein is inducible in a phosphate-deficient medium and then represents a very high percentage of the total synthesized proteins.

Hyperproduction obtained after cloning of the gene
PhoS in a multicopy vector is not lethal to the cell.

 Finally, this protein contains no methionine outside the signal sequence cleaved during export, this is of great interest in the particular application that is made below.

 Indeed, the aforementioned sequence makes it possible to prepare a fused protein containing the product of the PhoS gene fused with the product of the X gene, that is to say the desired protein. This fused protein is exported to the periplasmic space and can therefore be recovered by osmotic shock. Since it comprises, in addition, according to the preceding structure, a cleavage site between the two protein elements, it is possible to perform this cleavage and thereby recover the X protein.

 Although it is preferred to use the complete PhoS gene, it is nevertheless possible to use only a part of this gene, in particular it is possible to use the PhoS gene truncated at a portion of its 3 'end.

 In general, it will be preferable to truncate the 3 'end by only a few codons when this is necessary to obtain particular C-terminal structures.

 Of course, the PhoS gene must not have a stop codon, so as to allow the fusion with the protein X.

 As regards the Cl sequence of cleavage, it may be a coding sequence for one or more amino acids that can be cleaved chemically or enzymatically. The simplest cleavage site is obviously, for the moment, the ATG sequence corresponding to the start codon of the X gene which codes for methionine, which can be cleaved using cyanogen bromide.

 This cleavage must be able to take place only between the two fused proteins, this implies that the X gene does not have a coding sequence for methionine. This can be achieved by performing, for certain genes, point mutations by techniques which are known to those skilled in the art, in particular by mutations using synthetic oligonucleotides.

 The following examples were carried out with the GRF gene, but it is of course possible to use other genes coding for proteins of industrial interest.

 The invention relates to E. coli, optionally phos, transformed by the vectors described above, and a protein preparation process in which the E. coli strains are cultured. coli transformed and the fused protein is recovered in the osmotic shock supernatant, which protein is then cleaved at the cleavage site (Sc).

 It is advantageous to add phenylmethanesulfonyl fluoride (PMSF) at the concentration of 2 mM in the culture medium 2 hours after transfer of the bacteria on phosphate limiting medium. This helps to considerably improve the stability of the fusion protein. PMSF is then used in the purification steps.

 A particularly interesting method for the separation of the fused protein is to use an immunoabsorbent column to which an anti-PhoS antibody is attached. This makes it easy to separate the fused protein from the medium to effect cleavage.

 The following examples are intended to highlight other advantages and features of the present invention but obviously do not limit it.

EXAMPLE 1
The model system used is the production of the tGrowth Hormone Release Factor (GRF) peptide containing 44 amino acids. The synthetic GRF gene was purified from plasmid 196.1. This gene codes for a protein
Modified GRF in which internal methionine No. 27 was replaced by leucine and methionine was introduced at the amino-terminus.

The plasmid 196.1 was first hydrolysed by
EcoRI, Klenow polymerase-treated to remove the adhesive end, then cut with SalI enzyme.

The GRF gene obtained was then introduced into the cloning multisite of the pUC12 vector (Vieira J. et al.
Messing J. (1982) Gene 19, 259-268) having 11 unique restriction sites coding in the 3 phases and thus allowing to keep the reading phase with the PhoS gene.

For this purpose, the pUC12 vector was hydrolysed with Klenow polymer-treated BamHI and then with the enzyme
Sall. The GRF gene was then introduced into puy12 using a ligase (Figure 1).

 Strains of E. coli C600 and JM83 were transformed with the resulting plasmid. The sequence of the fragment of the SmaI positive clone (labeled with 32p in 5 ') - HincII was verified by the Maxam-Gilbert technique.

Site-directed mutagenesis, intended to introduce a unique restriction site, just before the stop codon of the PhoS gene was then performed. For this purpose, the fragment
PstI-EcoRI vector pSN 5182 (Anemura M., Shinagawa A.,
Makino K., Otsuji N. and Nakata A. (1982) J. Bacteriol. 152, 692-701) was first introduced into the vector M13mp9 (Messing J. and Vieira J. (1982) Gene 19, 269-275) to obtain
RFR311. The single strand containing the fragment was then prepared.

Mutagenesis was then performed with an oligonucleotide as shown below

<tb> - <SEP> - <SEP> - <SEP> Tyr <SEP> - <SEP> - <SEP> - <SEP> Stop <SEP> - <SEP> - <SEP> - <SEP> Stop
<tb> - <SEP> - <SEP> - <SEP> TAC <SEP> TAA <SEP> TAA <SEP>) <SEP> (Sequence <SEP> PhoS)
<tb> - <SEP> - <SEP> - <SEP> TAC <SEP> TAA <SEP>) <SEP> (oligonucleotide)
<tb><SEP> Tire <SEP> t <SEP> Val <SEP> Stop
<tb><SEP> SnaBI
<Tb>
The selection process used is that shown in Figure 2.

 Several clones are thus obtained in which the first stop codon of PhoS has been replaced by the codon GTA (coding for valine) and a SnaBI site has been created.

 The mutated PstI-EcoRI restriction fragment was reinserted into plasmid pSN 5182. The SmaI-HincII fragment of pUC12 GRF, containing the synthetic gene for GRF, was then introduced into the SnaBI site of mutated pSN5182.

The following construction is thus obtained

<tb> Tyr <SEP> I <SEP> Gly <SEP><SEP> Asp <SEP> Phe <SEP> I <SEP> Met <SEP> - <SEP> GRF <SEP> - <SEP> Stop
<tb> TAC <SEP> I <SEP> GGG <SEP> GAT <SEP> CAA <SEP> TTC <SEP> ATG
<tb> PhoS
<tb><SEP> PHOS <SEP> phots <SEP> | <SEP> Gly <SEP> Asp <SEP> Glr, <SEP> Phe <SEP> Met
<tb><SEP> CNBr
<Tb>
CNBr cleavage of the fusion protein can then provide the GRF peptide.

This system is generalizable and can be applied to produce and export any peptide in the periplasm. The presence of a single methionine in position
NH2-terminal allows easy recovery of the peptide provided it does not contain internal methionine residues.

The plasmid 196.1 is obtained from the plasmid pBR322 by cloning the synthetic gene GRF (marketed by
Creative Biomolecules, 385 Oyster Point Blvd, South San
Francisco, CA 94080, USA) between the EcoRI and SalI sites.

The tS gene is described in the following articles: Surin et al. (1984) J. Bacteriol. 157, 772-788
Magota et al. (1984) J. Bacteriol. 157, 909-917.

Claims (11)

 1) DNA block intended for the export of a specific protein in E. coli, characterized in that it comprises at least the following sequence

 lacking a coding sequence similar to the sequence Sc.  to release the product of the X gene, X corresponds to the gene coding for the determined protein and  chemically or enzymatically cleavable amino acids  stop codon, - Sc corresponds to a coding sequence for one or more  ensuring its export, said gene being devoid of <tb> - PhoS is the PhoS gene or part of that gene <tb> Phots <SEP> Sc <SEP> X  2) Export vector characterized in that it is a plasmid which comprises an export block according to claim 1 and at least one origin of replication in E. coli.  3) Vector according to claim 2, characterized in that Sc is a coding sequence for methionine.  4) Vector according to claim 3, characterized in that Sc corresponds to an ATG codon including the start codon of the X gene.  5) Vector according to one of claims 2 to 4, characterized in that the X gene is the GRF peptide gene.  6) Strain of E. coli transformed with a vector according to one of claims 2 to 5.  7) Process for obtaining a specific protein characterized in that a strain of E. coli according to Claim 6 on a culture medium, in that the fused protein produced from the PhoS gene and the X gene is recovered from the supernatant of the osmotic shock and in that this fused protein is cleaved to recover the determined protein. .  8) Process according to claim 7, characterized in that the fused protein is isolated by passage over an anti-phoS immunoabsorbent column which fixes the fused portion which is then cleaved.  9) Method according to one of claims 7 and 8, characterized in that, when Sc code for methionine, cleaves the fusion protein CNBr.  10) Method according to one of claims 7 to 9, characterized in that the culture medium is deficient in phosphate.  11) Process according to claim 10, characterized in that the phosphate-deficient culture medium contains phenylmethane-sulfonyl-fluoride (PMSF).