Patent Application
20060014250
PEPTIDE: includes any natural or synthetic compounds containing two or more amino acids. Therefore, it comprises proteins, glycoproteins, and protein fragments derived from pathogenic organisms such as viruses, bacteria, parasites and the like, or proteins isolated from normal or pathogenic tissues, such as cancerous cells. It also includes proteins and fragments thereof produced by recombinant means that has been associated or not with other peptides coding for tumoral, viral, bacterial or fungic epitopes for forming a fusion protein
POLYNUCLEOTIDE: any DNA, RNA sequence or molecule having one nucleotide or more, including nucleotide sequences encoding a complete gene. The term is intended to encompass all nucleic acids whether occuring naturally or non naturally in a particular cell, tissue or organism. This includes DNA and fragments thereof, RNA and fragments thereof, cDNAs and fragments thereof, expressed sequence tags, artificial sequences including randomized artificial sequences.
VECTOR: a self replicating RNA or DNA molecule which can be used to transfer an RNA or DNA segment from one organism to another. Vectors are particulaly useful for manipulating genetics constructs and differents vectors may have properties particularly appropriate to express proteins in a redipient during cloning procedures and may comprise different selectable markers. Bacterial plasmids are commonly used vectors.
Introduction
The present invention is useful for production of recombinant proteins in eubacterial hosts. A eubacterial host defective in the genes for Met-tRNAi transformylase and polypeptide deformylase is described which grows in minimal and complex nutrient media at 30° C., 37° C. and 42° C. with near wild-type rate. In this eubacterium protein synthesis does not require N-formyl methionine as the protein initiator, protein synthesis instead being initiated with unmodified methionine. The absence of peptides which retain N-formyl methionine in this eubacterium makes it particularly suited for the expression of recombinant proteins for pharmaceutical use.
The present invention relates to the use of a bacterial strain lacking of transformylase and deformylase activity for the production of non formylated peptides or proteins and also the selection of a mutant bacteria which is not spntaneousely reversible to the wild type for a formylase and deformylase acivities but has a doubling generation time similar to the growth rate of the wild type bacteria.
More particularly the invention provides the use of a bacterial strain lacking of deformylase and/or transformylase activities characterized in that it is non reversible for the deformylase and/or transformylase activities
The invention also provides the use of the bacterial strain according to the invention characterized in that it developes at a temperature of 37° C.
The invention further relates the use of a bacterial strain according to the invention characterized in that the bacteria is E. coli
The invention also provides bacteria strain lacking of the protein deformylation and/or transformylation activity characterized in that—said bacteria is not able to revert spontaneousely for these activities,—the growth rate of said bacteria is at least equivalent to he wild type growth rate.
The invention is also concerned with the use of the bacteria according to the invention for production of non formylated homologous or heterologous peptides or proteins.
The invention also relates to a preparation process of peptides or proteins comprising the following steps:
Another object of the invention concerns a process according to the invention in which the bacteria comprises a polynucleotide coding for a mutator allele and a process according the invention in which the bacteria mutator phenotype has been complemented or the mutator allele has been replaced by the, wid type allele.
The invention also concerned a purified polynucleotide comprising a gene coding for a mutator allele and contained in the bacteria strain according to the invention and a purified polynucleotide according to the invention in which the mutator phenotype has been complemented or the mutator allele ha been replaced by the wild type allele
The invention further relates to an E. coli bacterial γ2045culture deposited at the CNCM on Jul. 5, 2001 (accession number: I-2694)
Another aspect of the invention is directed to a process for the selection of mutants of a bacteria according to the invention characterized by the culture under continuous proliferation conditions of said mutants and separation of the mutant of interest from the static bacteria.
The invention is also concerned with a purified protein obtained after the expression of the heterologous or homologous insert of interest and deprived of formyl residue.
Other objects and advantages of the present invention will be apparent upon reading the following non restrictive detailed description made with reference to the accompanying drawing,
The present invention is useful for production of recombinant proteins in eubacterial hosts. A eubacterial host defective in the genes for Met-tRNAi transformylase and polypeptide deformylase is described which grows in minimal and complex nutrient media at 30° C., 37° C. and 42° C. with near wild-type rate. In this eubacterium protein synthesis does not require N-formyl methionine as the initiator methionine, protein synthesis instead is initiated with unmodified methionine. The absence of peptides which retain N-formyl methionine in this eubacterium makes it particularly suited for the expression of recombinant proteins for pharmaceutical use.
In eubacteria peptide synthesis is initiated at methionine start codons which are read by N-formyl methionine tRNA Prior to translation initiation the methionyl moiety of the charged tRNA is N-formylated by the action of Met-tRNAi transformylase (E.C.). The N-formyl group is removed from the native protein by polypeptide deformylase (E.C. 3.5.1-27), and the initiator methionine can then be cleaved off by methionine aminopeptidase, completing the primer methionine cycle. In contrast, archnea and eukaryotes have a primer methionine cycle devoid of N-formylating and deformylating activities (for review see Mazel et al., 1994, 1996).
Expression of eukaryotic proteins in eubacterial hosts often results in the production of recombinant proteins that retain an N-terminal formylmethionyl residue (examples include bovine sornatotropin [Bogosian et al., 1989]; ec1 growth hormone [Sugimoto et al., 1990]; human granulocyte colony-stimulating factor [Clogston et al., 1992]; bovine fatty acid-binding protein [Specht, et al., 1994]; bovine cytochrome P450 [Dong et al. 1995]; Methanothermus fervidus histone A [Sandman et al., 1995]; human interleukin-5 [Rose et al., 19921; human parathyroid hormone [Rabbani et al., 1988; Hogset et al., 1990]; human gamma-interferon [Honda et al., 1989]. In addition retention of N-formyl methionine has been found in endogeneous E. coli proteins (Hauschild-Rogat, 1968; Marasco et al., 1984; Milligan and Koshland, 1990). Since N-formylated peptides are a major indicator of eubacterial infections for the mammalian immune system ( ) and are highly immunogenic ( ), incomplete deformylation precludes, for example, the use of N-formylated preparations for therapeutic purposes. Several approaches to circumvent this problem have been proposed, e.g., expression in the presence of trimethoprim and thymidine (Sandman et al., 1995), overexpression of peptide deformylase in the host (2), expression as a protein fusion either with an N-terminal peptide that can be removed in vitro by a specific protease ( ) or with an N-terminal leader peptide which is cleaved during transport of the nascent protein in a non-cytoplasmatic compartment ( ). Finally, the N-formyl group may also be removed by mild acid hydrolysis ( ), or the fraction of the protein retaining N-formyl methionine may be separated from the correctly processed protein by purification procedures ( ).
Each of these approaches has significant disadvantages. Addition of trimethoprim and thymidine is costly, requires manipulation of the culture that will express the recombinant protein, and may slow down growth of the host. Overexpression of peptide deformylase requires a stable plasmid construct in the host that has to be selected for; moreover, deformylation may be less than 100% effective ( ). Expression of fusion proteins requires exact molecular constructions; chemical hydrolysis with acid may cause dammage to the rest of the protein. Finally, none of these approaches guarantees a final preparation that is absolutely free of N-formylated peptides derived either from the recombinant protein or from contaminations with endogeneous host peptides.
The present invention describes an E. coli strain with a primer methionine cycle similar to that in eucaryotic cells. This strain does non longer harbor the def-fmt operon encoding Met-tRNAi transformylase and polypeptide deformylase and thus can not N-formylate Met-tRNAi. Removal of N-formyl groups from expressed proteins by any of the techniques described above is thus no longer required.
The fmt and def genes from E. coli were previously isolated (Guillon et al., 1992; Mazel et al., 1994) and shown to be highly conserved among eubacteria (Mazel et al.,). Deletion mutants for either the fmt gene (Guillon et al., 1992) or the entire def-fmt operon (Mazel et al., 1994, D[def-fmt]) were created. The resulting mutants were reported to be severely impaired in growth. The fmt mutant has an 8.61-fold decreased growth rate at 37° C. in rich medium and does not grow at 42° C. (Guillon et al., 1992). The def-fmt mutant has a similarly decreased growth rate in minimal medium at 37° C., and growth is completely impaired in this medium at 42° C., ( ). Whereas deletion of the fmt gene alone leaves the mutant bacteria viable, deletion of the def gene alone as well as re-introduction of the fmt gene into a def-fmt background is lethal (Mazel et al., 1994), demonstrating that essential bacterial proteins either have to be deformylated, and/or that the initiator methionine has to be cleaved off in order to render these proteins functional.
For the purpose of the present invention, a def-fmt deletion mutant was selected for enhanced growth rates under permanent proliferation in minimal medium at 37° C. until its growth rate approximated that of the parent wild-type bacterium. For a second application, the def-fmt deletion mutant previously selected for growth at 37° C. was selected for enhanced growth rates under permanent proliferation in minimal medium at 42° C. until its growth rate approximated that of the parent wild-type bacterium.
1. Selection of enhanced growth rate in the def-fmt mutant (figure) 2. Growth rate of evolved strains at 30° C., 37° C. and 42° C. in minimal medium and rich medium (table)
An E. coli strain for expression of N-formyl-free polypeptides
In eubacteria peptide synthesis is initiated at methionine start codons which are read by N-formyl methionine tRNA. Prior to translation initiation the methionyl moiety of the charged tRNA is N-formylated by the action of Met-tRNAi transformylase. The N-formyl group is removed from the native protein by polypeptide deformylase, and the initiator methionine can then be cleaved off by methionine aminopeptidase, completing the primer methionine cycle (
Expression of eukaryotic proteins in eubacterial hosts often results in the production of recombinant proteins that retain an N-terminal formylmethionyl residue. Since N-formylated peptides are highly immunogenic, incomplete deformylation precludes, for example, their use for therapeutic purposes. Several approaches to circumvent this problem have been proposed, e.g., expression in the presence of trimethoprim and thymidine (1) or overexpression of peptide deformylase in the host (2).
The inventors have opted for a radical solution, simplifying the primer methionine cycle in Escherichia coli by deletion of the def-fmt operon that encodes polypeptide deformylase and met-tRNAi transformylase, and improving the resulting, crippled strain by selecting for increasing growth rates (and therefore improved rates of protein synthesis) under permanent proliferation in suspension.
The inventors have isolated the def and fmt genes from E. coli and created a deletion mutant (D[def-fmt]) devoid of both genes (3). The resulting strain was found to be viable, however its growth rate was dramatically reduced, from 0.9 per h to 0.25 per h in minimal medium at 37° C. Protein synthesis in living cells is dependent on the concerted action of a complex assembly of the protein and rRNA constituents of ribosomes and a host of factors catalyzing aminoacylation of tRNAs, initiation, elongation and termination of translation as well as maturation of nascent polypeptides. N-terminal formulation is among the most conserved features that distinguish eubacteria from archaea and eukaryotes. Removing the enzymes that catalyze the corresponding reactions is therefore expected to remove the efficiency of protein synthesis far from its wild-type optimum. Evolutionary ressurection from this type of genetic injury will require multiple adaptive mutations to render the bacterial translation machinery more similar to that found in eukaryotes. State-of-the-art technologies for directed evolution ex vivo are unable to predict and select the adaptive mutations that would re-establish wild-type protein synthesis rates in a D(def-fmt) background.
In vivo evolution of the D(def fmt) mutant under permanent proliferation in suspension in a turbidostat regime yields variants with increasing growth rate, approximating wild-type growth rate after about 1 month (ca. 300 generations) of permanent selection (
The evolutionary process can be accelerated by increasing variation in the population (
Current technology for continuous proliferation of cells in suspension suffers a major drawback, selection of adhesive variants which stick to inner surfaces of the device and escape the selective pressure imposed by continuous or conditional dilution (4). In principle, this can be avoided by serial subculture of cells in suspension (5), a technique where cells in suspension are frequently transferred into fresh culture vessels (i.e., surfaces are periodically discarded), creating a selective disadvantage for static variants. At an industrial scale, serial subculture technology has not been systematically exploited because it is laborious and requires absolute sterility during transfers.
| Number | Date | Country | |
|---|---|---|---|
| 60303065 | Jul 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10189505 | Jul 2002 | US |
| Child | 11176368 | Jul 2005 | US |
