MXPA00010057A - Novel constructs for controlled expression of recombinant proteins in prokaryotic cells - Google Patents
Novel constructs for controlled expression of recombinant proteins in prokaryotic cellsInfo
- Publication number
- MXPA00010057A MXPA00010057A MXPA/A/2000/010057A MXPA00010057A MXPA00010057A MX PA00010057 A MXPA00010057 A MX PA00010057A MX PA00010057 A MXPA00010057 A MX PA00010057A MX PA00010057 A MXPA00010057 A MX PA00010057A
- Authority
- MX
- Mexico
- Prior art keywords
- nucleic acid
- acid sequence
- promoter
- protein
- tryptophanase
- Prior art date
Links
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Abstract
The invention concerns a novel construct for expressing a gene coding for a recombinant protein of interest placed under the control of the Ptrp tryptophan operon promoter in a procaryotic host cell. The invention is characterised in that the construct comprises a nucleic sequence capable of inactivating the gene coding for a TnaA tryptophanase when said nucleic sequence is introduced in said host cell. The invention also concerns vectors containing said construct and host cells transformed by said vectors. The invention further concerns methods for producing said recombinant proteins using said novel constructs.
Description
NOVEDOS CONSTRUCTIONS FOR THE CONTROLLED EXPRESSION OF RECOMBINANT PROTEINS IN PROCEDURAL CELLS
DESCRIPTIVE MEMORY
The present invention comprises a novel construct for expressing a gene encoding a recombinant protein of interest placed under the control of the tryptophan operon promoter in a prokaryotic host cell, characterized in that the construct comprises a nucleic acid sequence that is capable of inactivating to the gene encoding a TnaA triptofanase when said nucleic acid sequence is introduced into said host cell, the vectors containing said construction and the host cells transformed with said vectors. One aspect of the invention are also methods for producing said recombinant proteins using these novel constructs. The present invention is generally used to produce recombinant polypeptides or proteins by so-called recombinant DNA methods. More particularly, the present invention relates to the production of polypeptides or recombinant proteins by transformed host cells which are of bacterial type, and in which the expression is directed by, or is under the control of, the operator / promoter of the operon of tryptophan Ptrp (Nichols &Yanofsky, 1983).
Escherichia coli (E. coli) is the organism most commonly used and best characterized for the purpose of production of recombinant proteins. Several expression systems are used in E. coli and, among them, the promoter of the tryptophan operon Ptrp is considered to be one of the strongest (Yansura &Bass, 1997). However, not all recombinant genes are expressed with the same efficacy by E. coli. It has been described and observed that the accumulation of a recombinant protein produced during the culture of transformed host cells can rapidly lead to instability of plasmids, a decrease in cell growth or even cessation thereof, and a decrease in the overall yield of the recombinant product . In this case, it is important to have a controlled and regulated expression system that makes it possible to divide the production process into two phases; a first phase called cell growth in which the activity of the promoter is minimal, followed by a phase called induction or derepression that favors the expression and accumulation of the recombinant protein. Ptrp, the promoter of the tryptophan operon of E. coli, is suitable for producing recombinant proteins due to its inducible nature. Repression at the operator level of Ptrp is carried out by the product of the regulatory trpR gene when this product, which is also called trp aporrepressor, binds to tryptophan (corepressor). The absence of tryptophan makes the TrpR protein incapable of binding to the operator, thus causing a derepression of the tryptophan operon. Various examples of expression of heterologous genes under the control of Ptrp show that the leakage of the expression thereof is too great to allow the production, under satisfactory conditions, of recombinant proteins, in particular those that are toxic to the cell (Yansura and Henner, 1990). The TrpR regulatory protein is subjected to a mechanism of self-regulation (Kelley &Yanofsky, 1982), and its concentration tends toward an average value of 120 molecules per K-12 cell of E. coli, in the presence of an excess of exogenous tryptophan ( Gunsalus, Gunsalus Miguel &Gunsalus, 1986). This concentration, although it is sufficient to correctly regulate the activity of the individual chromosomal promoter of Ptrp, may prove to be limiting against several dozens of vectors containing the same promoter. With respect to tryptophan, it can also be a limiting factor even if it is provided in excess in the culture medium. In E. coli there is, in fact, a triptophanase activity that is encoded by the tnaA gene, and which is capable of degrading tryptophan to indole, thus deviating from its regulatory function (Snell, 1975). In addition, tryptophanase can be induced by tryptophan, which makes any attempt to compensate for this phenomenon of degradation with an increase in the supply of tryptophan, be useless. Several procedures have been conceived and described aimed at obtaining the best possible control of the leakage of the expression. However, some have the disadvantage of being only applicable on a laboratory scale (Hasan &; Szybalski, 1995; Suter-Crazzolara & Unsicker, 1995), or to decrease the yield of the recombinant product (Stark, 1987). Accordingly, there is now a great need to develop a system for the controlled expression of recombinant proteins of interest which can be used on a large scale, and which makes it possible, in particular, to control the leakage of expression. This is precisely the objective of the present invention. The present invention relates to novel constructs based on the Ptrp expression system which, when introduced into a prokaryotic host cell, preferably of bacterial type, make it possible to decrease the residual expression of recombinant genes at the beginning of the culture, these novel constructions providing improved control of the synthesis of recombinant proteins. One aspect of the present invention is a construct for expressing a gene encoding a recombinant protein of interest placed under the control of the tryptophan operon promoter Ptrp in a priocarotic host cell, characterized in that the construct comprises a nucleic acid sequence that is capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell. The term "recombinant protein of interest" will be used to refer to all proteins, polypeptides or peptides that are obtained by genetic recombination, and which can be used in fields such as human or animal health, cosmetology, human or animal nutrition. , the agroindustry or the chemical industry. Among these proteins of interest may be mentioned in particular, but without being limited to the
• same, of: 5 - a cytokine, and in particular an interleukin, an interferon, a growth factor and a tissue necrosis factor, and in particular, a hematopoietic growth factor (G-CSF, GM-CSF), a hormone such as human growth hormone or insulin, a neuropeptide, - a factor or cofactor involved in coagulation and in
Particularly factor VIII, von Willebrand factor, antithrombin III, protein C, thrombin and hirudin, an enzyme and in particular trypsin, a ribonuclease and β-galactosidase, an inhibitor of a tai enzyme such as a1 -anti-trypsin and viral protease inhibitors, - a protein capable of inhibiting the initiation or progression of cancers, such as the expression products of tumor suppressor genes, for example the P53 gene, - a protein capable of stimulating an immune response or a antibody such as, for example, proteins, or their active fragments, or the outer membrane of a Gram negative bacterium, in particular Klebsiella OmpA proteins or the G protein of human respiratory syncytial virus, - a protein capable of inhibiting a viral infection or its development, for example the antigenic epitopes of the virus in question, or modified variants of viral proteins which can compete with native viral proteins, - a a protein that can be contained in a cosmetic composition, such as substance P or a superoxide dismutase, - a dietary protein, - an enzyme capable of directing the synthesis of chemical or biological compounds, or capable of degrading certain toxic chemical compounds, or alternatively, - any protein that is toxic with respect to the microorganism that produces it, in particular if this microorganism is the E. coli bacterium such as, for example, but not limited thereto, the protease of the HIV-1 virus, the ECP protein (ECP for cationic eosinophilic protein) or poliovirus proteins 2B and 3B. The expression "nucleic acid sequence capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell", will be used to refer to a nucleic acid sequence capable of modifying said gene in such a way that this modification leads to the loss of tryptophanase activity of said host cell, the expression product of said modified gene being unable to degrade the tryptophan to indole, and thus deviating from its regulatory function. Among said nucleic acid sequences capable of inactivating the gene encoding a TnaA tryptophanase when one of said nucleic acid sequences is introduced into said host cell, a nucleic acid sequence encoding a TnaA is preferred.
• inactivated tryptophanase obtained by mutation, such as by substitution, insertion and / or deletion of at least one nucleotide of the nucleic acid sequence encoding an active tryptophanase TnaA. The invention comprises a construction according to the invention, characterized in that the prokaryotic host cell is a Gram negative bacterium, preferably belonging to the E. coli species.
• The invention also relates to a construction according to the invention, characterized in that it also comprises, towards the 5 'end of said nucleic acid sequence capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell, all or part of the
The nucleic acid sequence of the promoter of the Ptna tryptophanase operon. Preferably, the invention relates to a construction of
• according to the invention, characterized in that said nucleic acid sequence capable of inactivating the gene encoding a TnaA triptofanase when said nucleic acid sequence is introduced into said cell
Host, comprises a mutated fragment of the coding sequence of said TnaA tryptophanase. Preferably, the invention relates to a construction according to the invention, characterized in that said mutated fragment is obtained by inserting a detection codon at a position such that the sequence of the mutated fragment obtained in this manner encodes a protein fragment that lacks activity of tryptophanase. Also preferably, the invention relates to a construct according to the invention, characterized in that said mutated fragment is a mutated fragment of the TnaA coding sequence of tryptophanase from said host cell. With respect to the nucleic acid sequence coding for the TnaA tryptophanase from E. coli, and its Ptna promoter, reference will be made in the present description to the sequence published by Deeley and Yanofsky (1981). With respect to the nucleic acid sequences encoding the operator / promoter of the tryptophan operon Ptrp, reference will be made to the sequence published by Yanofsky et al. (1981). The invention also relates to a construction according to the invention, characterized in that said nucleic acid sequence capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell, comprises an acid sequence nucleic acid comprising all or part of the sequence of a promoter which is followed, in position 3 ', by a nucleic acid sequence that encodes a molecule which is a ribonucleotide or protein in nature, and which acts negatively on the promoter Ptrp.
Preferably, the present invention relates to a construct according to the invention, characterized in that said promoter which is followed, in position 3 ', by a nucleic acid sequence that encodes a molecule which is a ribonucleotide or protein. in nature, and which acts negatively on the Ptrp promoter, is all or part of the promoter of the Ptna tryptophanase operon from E. coli. Also preferably, the invention comprises a construction according to the invention, characterized in that said nucleic acid sequence encoding a molecule which is a
• ribonucleotide or protein in nature, and which negatively activates the Ptrp promoter, is the sequence encoding the epitopepressor of the tryptophan operon TrpR of E. coli, or one of its biologically active fragments such as that described by Gunsalus and Yanofsky (1980). The term "a nucleic acid sequence comprising
All or part of the sequence of a promoter "will be used to refer to a nucleic acid sequence comprising the entire sequence of a
• promoter, or one of its biologically active fragments, which is able to direct or control the expression of a gene which is functionally linked to it. In the present description, the expression "biologically active fragment of a promoter" will be used to refer to any sequence of a fragment of said promoter, whose fragment is capable of directing or controlling the expression of the gene which is located towards the end 3 'of said fragment, said gene being functionally linked to said fragment. In the present description, the expression "biologically active fragment of the tryptophan operon pill TrpR" will be used to refer to any fragment of said aporrepressant which has retained its repressor activity. In the expression "nucleic acid sequence encoding a molecule which is a ribonucleotide in nature and which acts negatively on the Ptrp promoter", the preferred ribonucleotides are those selected from the following sequences:
a) d'-AUUCGCGUCU ACGGCUUCAU CGUGUUGCGC-3 'b) 5'-AUUCGCGUCU ACGGCUUCAU CGUGUUGCGC AGCACAACGC GCCUGUCACC GGAUGUGUUU UCCGGUCUGA UGAGUCCGUG AGGACGAAAC AGG-3' c) d'-AUUCAGUACG AAAAUUGCUU UCAUAAUUCU AGAUACCCUU UUUACGUGAA CUU-3 'd) 5'- AUUCAGUACG AAAAUUGCUU UCAUAAUUCU AGAUACCCUU UUUACGUGAA CUUAGCACAA CGCGCGCUGUC ACCGGAUGUG UUUUCCGGUC UGAUGAGUCC GUGAGGACGA AACAGG-3 'e) 5'-AUUCGCGUCU ACGGCUUCAU CGUGUUGCGC AUUCAGUACG AAAAUUGCUU UCAUAAUUCU AGAUACCCUU UUUACGUGAA CUU-3' • f) d'-AUUCGCGUCU ACGGCUUCAU CGUGUUGCGC AUUCAGUACG AAAAUUGCUU UCAUAAUUCU AGAUACCCUU UUUACGUGAA CUUAGCACAA CGCGCCUGUC ACCGGAUGUG UUUUCCGGUC UGAUGAGUCC GUGAGGACGA AACAGG-3 'g) 5'-CUUCGCGUCC UGAUGAGUCC GUGAGGACGA AACGGCUUCC-3' • 10 h) d'-CUUCGCGUCC UGAUGAGUCC GUGAGGACGA AACGGCUUCC AGCACAACGC GCCUGUCACC GGAUGUGUUU UCCGGUCUGA UGAGUCCGUG AGGACGAAAC AGG-3 '.
Another aspect of the invention relates to a vector containing a construction according to the invention. Preferably, the vector according to the invention is characterized in that it is the vector pMAK705 [tnaAt] or the vector pMAK705 [Ptna :: trpR :: 3'tna]. The invention also relates to a prokaryotic host cell, preferably a bacterium of the species E. coli, transformed with a vector according to the invention.
In another aspect, an aspect of the invention is a method for producing a recombinant protein in a host cell using a construct according to the invention. An aspect of the invention is also a method for producing d a recombinant protein of interest according to the invention, wherein said construct is introduced into the DNA of the prokaryotic host cell. Preference is given to a method for producing recombinant proteins according to the invention, wherein said construct 0 is introduced into the DNA of the prokaryotic host cell with a vector according to the invention, preferably in accordance with the chromosomal integration method described in Examples 1 or 2. An aspect of the invention is also a method for producing recombinant proteins according to the invention, wherein said d construction is introduced without any other element of DNA which would allow a selective advantage to be associated with it. Preferably, the invention comprises a method for producing a recombinant protein of interest according to the invention, wherein said construct is introduced into the locus of the E. coli tryptophanase operon, preferably at the locus of the tna gene, and better still in the locus of the tnaA gene.
Preference is given to a method for producing recombinant proteins of interest according to the invention, characterized in that it comprises the following steps: a) transforming a prokaryotic cell with a vector according to the invention, and integrating said construction into the DNA of said invention; host cell; b) transforming said prokaryotic cell with a vector containing a gene encoding said recombinant protein of interest; c) culturing said transformed cell in a culture medium that allows the expression of the recombinant protein; and d) recovering the recombinant protein from the culture medium or said transformed cell. An aspect of the invention is also a method for producing recombinant proteins of interest according to the invention, characterized in that said method also comprises, between steps a) and b) of the method described above, a step of resolution and selection. The invention also relates to a method for producing recombinant proteins of interest according to the invention, in which the control of the expression of the recombinant proteins before the induction of the Ptrp promoter is obtained by inducing said promoter which is followed, in position 3 ', by a nucleic acid sequence that encodes a molecule which is a ribonucleotide or protein in nature and which acts negatively on the Ptrp promoter according to the invention.
Finally, the invention also comprises a production method according to the invention, in which the induction of said promoter which is followed, in position 3 ', by a nucleic acid sequence coding for a molecule which is a ribonucleotide or protein in nature, and which acts negatively on the Ptrp promoter according to the invention, is obtained by any means that allows an inhibitory or activating effect to be exerted on said promoter. Preferably, the invention comprises a method of
• production in accordance with the invention, in which the induction of said promoter which is followed, in the 3 'position by a nucleic acid sequence encoding a molecule which is a ribonucleotide or protein in nature, and the which acts negatively on the Ptrp promoter according to the invention, is obtained: 1 da) by selecting a suitable carbon source in the culture medium; or • b) adding tryptophan to the culture medium; or by a combination of items a) and b). Construction and vector systems, cells
prokaryotic hosts transformed with said vectors, and the methods of the invention which were described above and which will be exemplified in the following examples, are within the context of the control of the production of recombinant proteins in prokaryotic cells.
They are suitable for the expression of homologous or heterologous genes placed towards the 3 'end of the Ptrp operator / promoter. Two mutants are described more particularly below to illustrate the invention. They have the names ICONE 100 and ICONE 200 (ICONE for improved cells for d overexpression and non-fleeting expression). The modifications introduced in the ICONE line have the following characteristics: 1) they are integrated into the host chromosome, 2) since they are generated using a site-directed mutagenesis technique, they are directed towards a single site in the DNA
(bacterial r 10, this site being completely known, since it is the tna operon located at 83 min in the physical map of the E. coli K-12 genome.) In this regard, the consequences for the host from one point are fully identified. In particular, the possibility that cryptic functions are reactivated after chromosomal integration, as assumed in the case of random mutagenesis, is excluded, 3) the technology used in these examples for chromosomal integration (Hamilton et al. (1989)), excludes the possibility that other sequences are inserted into the bacterial DNA. In particular, the mutants
do not have any gene for resistance to an antibiotic. If they are used on an industrial scale, they offer the producer and the legislator the guarantee that they will not have selective advantages in case of accidental spreading in the environment.
In accordance with one aspect of the invention, a first type of mutant or transformed cell called ICONE 100 is described, which possesses a mutation in the tnaA gene, which leads to loss of tryptophanase activity. The phenotype associated with this mutation is a lack of d degradation of tryptophan. This type of mutant, after transformation with a reporter vector and culture in a medium which conventionally promotes the activity of tryptophanase, turns out to be superior to the isolate from which it is derived in terms of control of the repression by tryptophan. According to another aspect of the invention, a second type of mutant called ICONE 200 is described, which possesses a cassette for expressing the trpR gene under the control of the Ptna triptofanase promoter, integrated into the locus of the tnaA gene. The use of the locus tna as a target for integration leads, in the host bacterium, to a loss of tryptophanase activity, which causes, as described above, the inability to convert tryptophan to indole. Furthermore, the presence of the cassette Ptna :: trpR in the chromosome confers in this novel trpR gene, the characteristics of Ptna, that is, sensitivity to catabolic pressure (Isaacs, Chao, Yanofsky &Saiser, 1994, Botsford &DeMoss, 1971) and induction by tryptophan (Stewart &Yanofsky, 198d). The last property constitutes an innovation in which the Ptrp promoter of the plasmid is controlled, at the level of the transcription, by a chromosomal promoter, Ptna, which is antagonistic thereto. Surprisingly, after transformation with an expression vector and culture in a fermentor, ICONE 200 turns out to be superior to the isolate from which it was derived in terms of control of the repression by tryptophan. Bacteria that have one of the mentioned characteristics
# are previously useful for the controlled production of recombinant molecules. Thus, one aspect of the present invention is also the use of said transformed bacteria in a method for producing recombinant proteins. In the following examples, the advantage provided by the two mutants is clearly demonstrated using Escherichia β-galactosidase
coli as the recombinant protein. Another aspect of the invention resides in the characteristics of the introduced mutations. They are totally defined, controlled from a genetic and biochemical point of view, directed to the tna locus of E. coli, and free of a selection marker. 1d The mutant or transformed microorganisms of the invention are constructed using prokaryotes, more precisely Gram-negative bacteria belonging to the species Escherichia coli. The promoter properties of the tryptophan operon from E. coli (which can be induced by tryptophan, sensitive to catabolic repression), were used to
to direct the transient synthesis of a mediator which acts negatively on the expression directed by Ptrp. However, it is known that other bacterial species, particularly those that colonize the intestinal tract of animals, are capable of synthesizing a tryptophanase which can be induced by tryptophan (Snell, 197d). Accordingly, other different strains of E. coli are suitable for carrying out the methods described and for producing recombinant proteins herein. # The following examples and figures are intended to illustrate the invention without limiting the scope of the invention in any way.
Legends of the figures: Figure 1: Growth kinetics of the strains RV308, ICONE 100 and ICONE 200 x pVA-ßgal. An OD of d80 nm corresponds to the measurement
• 10 of the optical density measured by spectrophotometry. Figure 2: Kinetics of β-galactosidase activity of strains RV308, ICONE 100 and ICONE 200 x pVA-ßgal. Bacteria transformed with a vector containing the β-galactosidase gene under the control of the Ptrp promoter are cultured in a fermentor. The β-1d galactosidase activity is measured by incubating a cell extract in the presence of ONPG (β-galactosidase specific substrate). Figure 3: Comparison of the growth kinetics of strains RV308 and ICONE 200 of E. coli transformed with the vector pVA-polio2B. Figures 4A and 4B: Immunoblot in the intracellular extracts of 20 cultures of RV308 and ICONE 200 transformed with the vector pVA-polio2B. Figure 4A: RV308 x pVA-polio2B Figure 4B: ICONE 200 x pVA-polio2B Figure d: Analysis by SDS-PAGE of purified polio2B protein by nickel affinity chromatography. A: Experiment No. 2: induction with d μg / ml of lAA when the
• optical density is equal to 32.d; d B: Experiment No. 1: induction with 2d μg / ml of lAA when the optical density is equal to 32.d; C: Experiment No. 4: induction with d μg / ml of lAA when the optical density equals 63. d; D: Experiment No. 3: induction with 25 μg / ml of lAA when the optical density equals 62. d; MW: molecular weight (kDa) of the marker. Figure 6: growth kinetics of ICONE 200 x pVA-polio2B: influence of the induction time and the concentration of the inducer. M Experiment No. 1: induction with 2d μg / ml of lAA when the optical density is equal to 32.5; D Experiment No. 2: induction with 5 μg / ml of lAA when the optical density is equal to 32.5; • Experiment No. 3: induction with 2d μg / ml of lAA when the optical density is equal to 62. d; 20 O Experiment No. 4: induction with 5 μg / ml of lAA when the optical density is equal to 63.5. The arrows indicate the moment of induction.
The invention is based on the stable introduction of mutations in the genome of the host strain. All the modifications given in the following examples are introduced into the tna locus of E. coli, consisting schematically of the following series: A) Ptna promoter, B) coding sequence of the tnaA (tryptophanase) gene, C) intergenic region, D) coding sequence of the tnaB gene (tryptophan permease),
E) Transcription terminator. More specifically, the modifications refer to element (B). This is replaced in favor of an element (b), whose characteristics in various constructions are the following:
TABLE 1 Nature of mutations carried by ICONE 100 and ICONE 200
EXAMPLE 1
Construction of the mutant ICONE 100
A DNA fragment, labeled as tnaAT, is amplified by PCR. It goes from position -276 to position +1064 with respect to the first nucleotide of the tnaA coding sequence. This fragment, which overlaps the Ptna and tnaA promoter, is amplified by two-part PCR. Part I goes from position -275 to position +220. It is amplified with the help of the oligonucleotides Trpd (sense) and Trp2 (antisense), whose
• 10 sequence is:
Trpd: 5'-CGGGATCCGTGTGACCTCAAAATGGTT-3 'BamHI Trp2: 3'-CTACGCGCCGCTGCTTCGGATTAGATCTCG-5' (antisense) Detention Xbal Part II is localized in the tnaA coding sequence,
• immediately 3 'from part I. It goes from position +221 to position +1064. It is amplified with the help of the Trp3 and Trp4 oligonucleotides:
Trp3: d-CGTCTAGACAGCGGCAGTCGTAGCTAC-3 '20 Xbal Trp4: 3'-CCTTCTCTAACCGCAACAGTTCGAACG-d' (antisense) Hindlll PCR reactions are carried out using as matrix the E. coli K-12 colonies used in the pH regulator of Taq polymerase (AmpliTaq Gold CETUS, USA). The amplification products are precipitated with ethanol, and then digested with the appropriate restriction enzymes (BamHI / Xbal for part I, Xbal / Hindlll for part II). An analysis of agarose gel stained with ETB makes it possible to verify that the fragments have the expected size (Deeley &Yanofsky, 1981). The tnaAT fragment is generated by ligating the two fragments I and II at the Xbal site. It differs from the natural sequence by the presence of a stop codon at position +221, followed by a Xbal restriction site. This fragment tnaAT is cloned into the vector pRIT28 (Hultman, Stahl, Hornes &Uhlen, 1989), at the BamHI / HindIII sites, and sequenced. The tnaAT fragment is subcloned into the pMAK70d vector (Hamilton, Aldea, Washbum, Babitzke &Kushner, 1989), giving pMAK70d [tnaAT]. The method used to generate a genetic rearrangement in E. coli is that described by Hamilton et al. (1989). It is based on the use of the suicide vector pMAK70d, which has a heat-sensitive origin of replication that is functional at 30 ° C, but inactive beyond 42 ° C, and the chloramphenicol resistance gene (CMP). RV308 from E. coli (Maurer, Meyer &Ptashne, 1980) is transformed with 4 μg of vector pMAK70d [tnaAT], and the transformation mixture is deposited on plates containing LB medium + 20 μg / ml of CMP. After incubation overnight at 30 ° C, three clones are subcultured in liquid medium of LB + 20 μg / ml of CMP, and incubated at 30 ° C with shaking, until an OD of 680 nm close to 1 is reached. The suspensions are then deposited in LB medium + 20 μg / ml of CMP, and incubated at 44 ° C and at 30 ° C. The colonies that develop at 44 ° C (= cointegrants) have a chromosomal integration of the vector, this integration being promoted by the existence of sequence homologies between the chromosome and the insertion that the vector possesses. The so-called resolution phase consists of promoting the excision of the vector by means of a recombination mechanism between repeated frequencies present in the chromosome. Some clones isolated at 44 ° C are cultured in liquid medium of LB + 20 μg / ml of CMP at 30 ° C for three days, renewing the medium regularly. The suspensions are then diluted, deposited on LB agar medium + 20 μg / ml of CMP, and incubated at 30 ° C until separate colonies appear. Several dozen colonies are subcultured in duplicate on LB agar medium + 20 μg / ml CMP at 30 ° C and 44 ° C. Colonies that do not develop at 44 ° C are selected by PCR, which indicates whether the resolution of the vector has retained the stop codon and the Xbal site at the tna locus. The oligonucleotides used are Trp6 (sense) and Trp7 (antisense), which are homologous to the desired mutation, and to a portion of the tnaA terminator, respectively:
Trp6: 5'-CGACGAAGCCTAATCTAGA-3 'Xbal of arrest Trp7: 3'-CCGATATTCCTACAATCGG-d'
• (antisense)
From 18 selected clones, 9 give an amplification fragment with the expected size, indicating the presence of the stop codon followed by the Xbal site in the tnaA gene. The other 9 clones do not give an amplification product, perhaps because the resolution step has
• 10 restored to the chromosome the non-mutated tnaA gene. Among the 9 positive clones, 4 are sampled and subjected to plasmid clearance by successive subculture in the absence of selection pressure. After cultivation for a few days, clones are obtained which again have become sensitive to chloramphenicol. The presence of mutation of inactivation of tnaA is confirmed in two different ways: firstly, a PCR amplification with the help of the oligonucleotides Trpd and Trp4, followed by digestion with Xbal, shows that the restriction site, which is lacking in RV308 from E. coli, is present in the tnaA gene of the mutants; then, cultivating the mutants in
a medium rich in tryptophan, followed by the indole test (adding Kovacs reagent to the culture medium), it is shown that the mutants have not generated idol, while the RV308 strain of origin produces indole under the same conditions. It follows then that the introduced mutation leads to loss of tryptophanase activity. A clone is selected for conservation purposes
• frozen. His name is ICONE 100.
EXAMPLE 2 Construction of the mutant ICONE 200
A fragment of DNA is constructed in vitro by
• 10 PCR amplification of three subunits. The first subunit located in the Ptna promoter goes from position -511 to position +3 with respect to the first nucleotide of the tnaA coding sequence. It is amplified using the oligo-nucleotides TrpR1 (biotinylated at the 5 'position) and TrpR2: 1 d TrpR1: d'-CTGGATCCCTGTCAGATGCGCTTCGC-3' BamHI TrpR2: 3, -CTTCCTAATACATTACCGGGTTG-5 '(antisense) The second subunit corresponds to the sequence of coding of the trpR gene of E. coli. It is amplified using the oligonucleotides TrpR3 and TrpR4 (biotinylated in the 6 'position): TrpR3: d'-GTAATGGCCCAACAATCACC-S' Start TrpR4: 3'-CACAACGACTTTTCGCTAACTGACGTCAG-5 '(antisense) Pstl The third subunit corresponds to the sequence located immediately 3 'of the tnaA coding sequence. It contains the intergenic region of the tna operon and a portion of the tnaB gene that codes for tryptophan permease. It is amplified using the oligonucleotides TrpRd and TrpRd: TrpRd: 5'-CGCTGCAGTTAATACTACAGAGTGG-3 'Pstl • 10 TrpR6: 3'-CCAGCTAATGAGGTAAGTTGGAAC-5' (antisense) Hindlll
The amplified fragments are purified according to the GeneClean method (Biol 01, Jolla, CA, USA). 16 Subunits I and II are merged as follows. In two separate tubes, each subunit is incubated with 30 μl of beads
• marked with streptavidin (Dynabeads, DYNAL, Norway). After 20 minutes at 37 ° C and d minutes at room temperature, the ligated DNA is denatured with 60 μl of NaOH at 0.1d M. The DNA molecules of
The single chain recovered in each supernatant are mixed in equal parts, and subjected to a hybridization reaction and an extension reaction in the presence of Taq polymerase (AmpliTaq Gold, CETUS, USA) in accordance with five PCR cycles. The reaction product is amplified by PCR with the aid of the oligonucleotides TrpR1 and TrpR4. The amplification product purified by the GeneClean method is digested with BamHI and PstI. The fragment isolated from this manner is cloned into the pRIT28 vector to give pRIT28 [Ptna :: trpR], and then sequenced. The lll subunit is digested with the enzymes Pstl and Hindlll, then cloned into pRIT28 to give pRIT28 [3'tna], and then the sequence is verified by DNA sequencing (ABI 373A, Perkin Elmer, USA).
• 10 The vector pRIT28 [Ptna :: trpR] is digested with the enzymes Pstl and
Hindlll, then ligated in the presence of subunit III, and isolated by itself from pRIT28 [3'tna] by double digestion with PstI / HindIII. The resulting vector is called pRIT28 [Ptna :: trpR :: 3'tna]. The insert is transferred into pMAK70d after double digestion with the BamHI and 15 Hindlll enzymes. The resulting plasmid is designated pMAK70d [Ptna :: trpR :: 3'tna]. The integration of the Ptna :: trpR :: 3'tna fusion in the tna locus of
• RV308 from E. coli is carried out under conditions similar to those described in example 1. For a short time, the strain is transformed with the vector pMAK70d [Ptna :: trpR :: 3'tna], and then subjected to the integration steps and
resolution. The selection of the colonies at the end of the resolution uses conditions that are slightly different from those used in example 1. The tna locus is amplified by PCR using the oligonucleotides TrpR11 and TrpR7: TrpR11: d'-GGGCAGGTGAACTGCTGGCG-3 'TrpR7: 3 , -GGTGCCGTTATAAGGGTCGGAC-d, (antisense) TrpR11 hybridized with the Ptna sequence towards the d 'end of TrpR1, and TrpR7 hybridized with the tnaB sequence towards the 3' end of TrpR6. The amplification product has a different size, depending on whether the gene set towards the 3 'end of Ptna is tnaA (situation found in RV308) or trpR (desired situation in the mutants). A colony that possesses trpR at the tna locus, is conserved and called ICONE 200. The analysis of its chromosomal sequence shows that it possesses the trpR gene immediately towards the 3 'end of the Ptna promoter. Culture in the presence of tryptophan confirms the absence of indole formation, which is a logical consequence of the loss of the tnaA gene.
EXAMPLE 3 Expression leak in the presence of succinate + tryptophan
This example describes the relative capabilities of RV308,
ICONE 100 and ICONE 200 from E. coli to control the expression of a recombinant protein under the control of the Ptrp promoter. For this purpose, an expression vector called pVA-ßgal was constructed, wherein the sequence coding for ß-galactosidase from E. coli is placed towards the 3 'end of the Ptrp promoter. The vector of origin used for this construction is pVAABP308 (Murby, Samuelsson, Nguyen, et al., 1995). Each of the three strains is transformed into the pVA-β-gal vector. The obtained transformants are grown individually in a complete medium (30 g / l of tryptic soy broth (DIFCO), 5 g / l of yeast extract (DIFCO)) overnight at 37 ° C. An aliquot of these previous cultures is transferred to 60 ml of M9.YE.SUCC medium (1X M9 salt solution (DIFCO), 5 g / l of yeast extract (DIFCO), 20 g / l of sodium succinate). After an incubation time at 37 ° C that allows reaching the exponential growth phase, a sample of each culture is removed. Growth is assessed by optical density at 50 nm of the bacterial suspension. The level of β-galactosidase activity is measured in each cell pellet. For this, 1 ml of culture is centrifuged for 3 minutes at 12000 g. The cell pellet is absorbed in 900 μl of pH buffer (60 mM Tris-HCl, pH 7.6 / 1 mM EDTA / 100 mM NaCl / 400 μg / ml lysozyme) and incubated for 15 minutes at 37 ° C. 100 μl of SDS (1% in 50 mM Tris-HCl, pH 7.5) are added, and the sample is placed at room temperature for 5 minutes. The test is carried out by mixing 30 μl of the sample, 204 μl of pH buffer (60 mM Tris-HCl, pH 7.6 / 1 mM MgCl2) and 66 μl of ONPG (4 mg / ml in 60 mM Tris-HCl, pH 7.d). The reaction mixture is incubated at 37 ° C. The reaction is stopped by adding 500 μl of 1 M Na 2 CO 3. The OD at 420 nm, related to the incubation period, is proportional to the activity of β-galactosidase present in the sample. As it is already known that E. coli RV308 has a complete deletion of the lac operon (Maurer, Meyer &Ptashne, 1980), the β-galactosidase activity tested is solely due to the expression of the gene brought to
• carried out by the vector pVA-ßgal. Table 2 summarizes the results obtained with each of the strains RV308, ICONE 100 and ICONE 200.
TABLE 2 Growth of strains RV308, ICONE 100 and ICONE 200 v leakage
• 10 expression (mean and standard error on the three experiments)
The results shown in table 2 show that the
mutants of the ICONE line develop at least as well as strain RV308 from which they are derived. The mutations introduced from this
• way they do not have negative effects on growth. Additionally, the β-galactosidase activity measured is different in the three strains. ICONE 100 has an intracellular activity that is approximately 4.d times
less than that of RV308. Under conditions of "succinate as a carbon source", where the activity of the Ptna promoter is at maximum (Botsford &DeMoss, 1971), the deletion of the tryptophanase gene in this manner leads to a decrease in expression leakage, probably limiting the degradation of intracellular tryptophan (corepressor). Under the same conditions, the degree of leakage of expression in ICONE 200 decreases further by approximately 10-fold with respect to ICONE 100. The activity of the plasmid Ptrp promoter is thus at a minimum in ICONE 200. First, the loss The activity of tryptophanase gives the bacterium the possibility of better controlling Ptrp as demonstrated for ICONE 100. However, ICONE 200 has a second property that distinguishes it from ICONE 100 in genetic terms and gives it, at an experimental level, a Additional advantage in terms of expression control. In this way, under
• 10 conditions where Ptna is active, ICONE 200 has the possibility of directing the overexpression of the TprR aporrepressor, and consequently, the leakage of expression measured at the level of the plasmid Ptrp promoter decreases by a factor that is close to 60 with respect to strain of origin RV308.
EXAMPLE 4 Expression leak in the presence of glycerol + tryptophan
• This example demonstrates the utility that ICONE 200 mutant provides in a fermentation culture medium and under fermentation conditions that are close to those that can be used industrially to produce recombinant proteins with the Ptrp system. Each of the three strains RV308, ICONE 100 and ICONE 200 is transformed with the vector pVA-ßgal. The transformants obtained are individually cultured in 200 ml of complete medium (30 g / l soy triptych broth (DIFCO), 5 g / l yeast extract (DIFCO)) overnight at 37 ° C. The cell suspension obtained is transferred in a sterile manner to
• one fermenter (model CF300 of Chemap, with capacity of 3.d I) d containing 1.8 liters of the following medium (concentrations for 2 liters of final culture): 90 g / l of glycerol, dg / l of (NH4) 2SO4, 6 g / l of KH2PO4, 4 g / l of K2HPO4, 9 g / l of Na3-citrate.2H2O, 2 g / l of MgSO4.7H2O, 1 g / l of yeast extract, 30 mg / l of CaCl2. 2H2O, 8 mg / l of ZnSO4.7H2O, 7 mg / l of CoCl2.6H2O, 7 mg / l of Na2MoO4.2H2O, 10 mg / l of MnSO4.1 H2O, 2 mg / l of
• 10 H3BO3, 8 mg / l CuSO4.5H2O, 64 mg / l FeCl3.6H2, 0.06% antifoam agent, 8 mg / l tetracycline and 200 mg / l tryptophan. The pH is adjusted to 7.0 by adding aqueous ammonia. The content of dissolved oxygen is maintained at 3% saturation by automatic regulation of the stirring speed and subsequently the ventilation flow rate,
measuring the dissolved O2. When the optical density of the culture reaches a value between 40 and 45, the induction is carried out by adding 25 mg / l of indole acrylic acid (SIGMA). An analysis is carried out by kinetics of the optical density of the culture (OD at 580 nm of the suspension) and intracellular activity
of β-galactosidase (see example 3). Figures 1 and 2 illustrate, respectively, the growth kinetics and the kinetics of the β-galactosidase activity of the three cultures.
The data provided in Figure 1 confirm the observation in Example 3: all three strains have comparable growth kinetics. The mutants of the ICONE line, from this point of view, have• retained the growth potential of E. coli RV308, and therefore
remain as potential candidates for industrial use. The data in Figure 2 show the impact of the mutations carried out by the ICONE strains on the expression of β-galactosidase in a fermentor. Clearly, in a medium based on glycerol, the presence or absence of tryptophanase activity has no effect on the control of the
• 10 expression, as certified by the first part of the curves of RV308 and ICONE 100, although it was observed that the exogenous tryptophan disappears more rapidly in the culture of RV308 than in that of ICONE 100 (data not shown). On the other hand, the ICONE 200 mutant exhibits better capacities to control the expression at the beginning of the culture: the activity of β-15 galactosidase remains low during the first 18 hours of culture, and later it begins to increase from t = to 20 h, the moment when the concentration of extracellular tryptophan becomes 0 (no data shown). The second part of the curve referring to ICONE 200 shows that the activity of β-galactosidase increases in a uniform manner so that it reaches a
level at the end of the crop that is close to that obtained with RV308. In this regard, it was shown that the regulatory system present in ICONE 200 provides a transient control of the plasmid Ptrp promoter. This control, exerted by the tryptophan and / or the carbon source, becomes ineffective in the second part of the culture and does not act against the maximum expression of the recombinant protein.
EXAMPLE 5 Control of the expression of a toxic protein
This example describes the behavior of strains RV308 and ICONE 200 in culture when they are transformed with a vector that transports, towards the 3 'end of the tryptophan promoter, the gene of a toxic protein. By way of example, and in a manner that illustrates the invention, the gene of interest is that which codes for the poliovirus 2B protein. It has been described that overexpression of this protein modifies the permeability of the membrane in the bacteria (Lama et al., 1992) and in eukaryotic cells (Aldabe et al., 1996), which makes a model of choice to study the consequences of the leakage of expression in E. coli. The gene coding for protein 2B is amplified from the vector pET3.2B (Lama et al., 1992) by a PCR reaction with the help of the following oligonucleotides: PO2.1 5'-GCGAATTCTGGCATCACCAATTACATAG-3 '(sense) EcoRI PO2.21 5'-GCAAGCTTAGTGGTGGTGGTGGTGGTGTTGCTTGATGACATAA
(antisense) HindIII GGTATC-3 'The amplification product is then digested with the restriction enzymes EcoRI and HindIII and subsequently cloned into an expression vector which is derived from pBR322 and which transports the PTPP promoter from tryptophan. The resulting vector, called pVA-polio2B, carries a sequence encoding the 2B protein fused, at its C-terminal end, to a poly (His) tail, under the control of the Ptrp promoter. The vector pVA-polio2B is introduced into the bacterium E. coli RV308 and ICONE 200 by transformation. A recombinant clone of each construct is subsequently cultured under conditions that are similar to those described in Example 4. The growth kinetics of the RV308 and ICONE 200 bacteria measured by the optical density at 580 nm are given in Figure 3. You can clearly see in this one that RV308 exhibits a considerable growth delay: the average generation time in the fermenter during the first 14 hours of cultivation is 1 hour 45 minutes, again only 1 hour 17 minutes for ICONE 200. After a culture during 24 hours, the optical density of strain RV308 is only equal to 13. Surprisingly, strain ICONE 200 reaches an optical density equal to 37 after 17 hours 30 minutes of culture, at which time the induction is carried out by addition of indole acrylic acid (lAA) at 25 μg / ml. The effect of induction is immediate: the rate of oxygen consumption drops abruptly (no data are shown) and growth stops.
Samples were taken at various periods of cultures, and analyzed for their recombinant protein content. Samples of biomass centrifuged at 8000 g are absorbed in a P1 pH regulator (25 mM Tris, 1.15 mM EDTA, 1 mg / ml lysozyme, pH 8) in a ratio of 5 d ml per 1 g of biomass. The biomass is resuspended, incubated for 16 minutes at room temperature, and then subjected to sound treatment for 2 minutes. The lysate is centrifuged again (10 000 g, 15 min., 4 ° C) so as to give a soluble fraction (supernatant) and an insoluble fraction (pellet absorbed in 200 μl pH regulator P2: 25 mM Tris, 1 mM 0 EDTA, pH 8). These samples are loaded on polyacrylamide gel and subjected to electrophoresis under denaturing conditions (SDS-PAGE). Then the gel is transferred to the membrane in accordance with the Westem-blot technique to reveal the presence of the recombinant protein. The antibody used is anti-poly (His) coupled to monoclonal peroxidase d (Sigma). The disclosure is carried out chemiluminescence with the ECL + kit (Amersham). Figures 4A and 4B give the result of immunoblots on the insoluble fractions derived from the RV308 and ICONE 200 cultures, respectively. Figure 4A shows that the recombinant protein is present in all the samples, ie from the start of the culture until the fermentation time t = 24h, even though no induction with lAA has been carried out. In contrast, with ICONE 200, no recombinant protein was detected before induction (Figure 4B). It is only after the lAA induction that protein 2B can be detected (in the insoluble fraction) and that the manifestation of its toxic nature is observed. In this way, these results demonstrate that ICONE 200 mutant has a clear utility with respect to strain RV308 from which it is derived, and makes it possible to produce an effective expression control in a fermentor.
EXAMPLE 6 Production of a toxic protein
This example is performed to demonstrate that a toxic protein or 0 can be expressed in an ICONE 200 culture of E. coli at a high cell density and under culture conditions that are suitable for industrial extrapolation. For this purpose the strain ICONE 200 of
E. coli transformed with the pVA-polio 2B vector. The results obtained in Example 5 indicate that the induction conditions should be optimized d in case the instantaneous growth decreases and then the bacterial lysis, caused by the expression, should be avoided. In this way, this example describes various tests created to optimize the production of recombinant protein per unit volume fermented by adjusting the concentration of the inducer and cell density in the induction. The culture conditions used are those described in example 4. The experimental combinations tested are those indicated:
Experiment No. 1: induction with 2d μg / ml of lAA when the optical density is between 30 and 36; Experiment No. 2: induction with 5 μg / ml of lAA when the
• optical density is between 30 and 3d; 5 Experiment No. 3: induction with 2d μg / ml of lAA when the optical density is between 60 and 65; Experiment No. 4: induction with 5 μg / ml of lAA when the optical density is between 60 and 65. For each experiment, samples of biomass in
F 10 several times after induction and analyzed according to the following protocol. Approximately 20 grams of biomass are absorbed in 100 ml of 1X starting pH buffer (prepared from an 8X concentrate: 1.42 g of Na2HPO42H2O, 1.11 g of NaH2PO4H2O, 23.38 g of NaCl, 100 ml, pH 7.4). The suspension is subjected to treatment with
sound for 3 x 5 minutes, and then centrifuged for 30 minutes at 20,000 g and 4 ° C. The pellet is absorbed in 15 ml of the start pH buffer + 6 M guanidine-HCl + 0.1% SB3-14 (N-tetradecyl-N, N-dimethyl-3-ammonium-1-propanesulfonate, Sigma), and Subsequently, it is incubated on ice for 1 hour. The suspension is centrifuged for 1 hour at 30,000 g and
4 ° C. The supernatant is filtered through 0.45 μ with the aim of purifying it by chelated metal affinity chromatography. A column that contains
1 ml of gel (HiTrap chelator, Amersham Pharmacia Biotech) is loaded with 1 ml of 0.1 M NiSO4, washed with 5 ml of water, and then equilibrated with 30 ml of pH buffer + 6M guanidine-CHI + 0.1 % of SB3- 14. The sample is then loaded into the column. A rinse with 60 ml of wash pH regulator (start pH buffer + 6M guanidine-HCl + 0.1% SB3-14 + 20mM midazole) makes it possible to remove most of the 5 proteins bound by nonspecific interactions. The recombinant polio-2B protein is eluted with 10 x 1 ml of elution pH buffer (pH buffer of start + 6M guanidine-HCl + 0.1% SB3-14 + 300mM imidazole). Fractions with the highest protein concentrations are pooled and subsequently desalted on Sephadex G-2d gel (PD10 columns,
• 10 Amersham Pharmacia Biotech). The quality and quantity of the polio-2B protein obtained in this way are estimated by electrophoresis under denaturing conditions (SDS-PAGE) and by testing the total proteins (BCA method, Pierce), respectively. Figure 5 shows an SDS gel with Coomassie blue staining
of the polio-2B proteins extracted and purified subsequently to the experiments 1 to 4 described above. The size of the protein
• Recombinant corresponds to the predicted theoretical size (11 kDa) of its coding sequence. In addition, it corresponds to the size of the highest protein observed in a Western blot after induction, in the ICONE lysate
200 of E. coli x pVApolio-2B (Figure 4B). Thus, it is likely that the proteins visible in Figure 5 correspond to the poliovirus 2B protein fused to a polyhistidine tail. In addition, the quality of the proteins obtained is identical under all conditions of induction under test.
Table 3 below summarizes the results obtained by combining several factors such as optical density upon induction, inductor concentration and culture time after induction.
TABLE 3 Influence of optical density on induction, of the concentration of the inducer and the time after induction in the production of expression of a toxic protein (example of polio-2B)
When comparing the groups of experiments (1-2) and (3-4), it is observed that the later the induction is carried out, the greater the production of expression. This confirms the utility of growing the biomass as much as possible before activating the induction. In the case of the experiments (3-4), approximately 70% of the carbon substrate is consumed at the time when the expression of the polio-2B protein is activated. With a strain such as ICONE 200, the cell growth part and the expression phase are completely separated, which makes it possible to optimize the production of the recombinant protein, even when this protein is toxic. In parallel with the induction time, the concentration of the inductor is also a parameter of influence. The best expression result obtained in this example corresponds to experiment number 4, where the concentration of the inducer related to the number of cells is the lowest (5 mg / l of lAA for a culture with an optical density equal to 63.5). Also in this experiment the toxic effect of polio-2B expression is the least noticeable, since the culture continues to develop after induction, while the growth stops completely in the other experiments (see figure 6). This is why it is particularly important to adjust the conditions of induction of a toxic protein so that optimum conditions are found between an inductor concentration that is too low to give an important expression and a concentration that is too high, causing the immediate suspension of the metabolism of the bacteria. When comparing the results of experiments number 4 and number 1, it is observed that an induction that is posterior (DO = 63.5 in comparison with 32.5) and less strong (concentration lAA equal to 5 mg / l compared to 25 mg / i) makes it possible to multiply by 3 to 4 times the amount of protein recombinant obtained per unit volume fermented. The E. coli strain ICONE 200, obtained by genetic modification according to the invention of a strain of industrial value, makes it possible to strictly control the expression of any gene placed in a plasmid vector towards the 3 'end of the tryptophan Ptrp promoter. This control is transient, since it is mediated by the exogenous tryptophan provided in the culture. The induction potential of Ptrp in ICONE 200 is conserved, and modulation via the lAA concentration is possible. Due to these properties, ICONE 200 allows the controlled expression of recombinant proteins under culture conditions that can be extrapolated on a large scale.
•
•
BIBLIOGRAPHY
Aldabe, R., Barco, A. & Carrasco, L., (1996). The Journal of Biolological Chemistry 271, 23134-23137. d Botsford, J.L. & DeMoss, R. D. (1971). Catabolite repression of tryptophanase in Escherichia coli. Journal of Bacteriology, 10d, 303-312. Deeley, M.C. & Yanofsky, C. (1981). Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12. Journal of Bacteriology, 147, 787-796. 0 Gunsalus, R.P., Yanofsky, C. (1980). Nucleotide sequence and expression of E, coli trpR, the structural gene for the trp aporepressor. Proceeding of the National Academy of Sciences, USA, 77, 12, 7117-7121. Gunsalus, R.P., Gunsalus Miguel, A. & Gunsalus, G.L. (1986). Intracellular Trp repressor levéis in Escherichia coli. Journal of Bacteriology, 5 167, 272-278. Hamilton, C.M., Aldea, M., Washbum, B.K., Babitzke, P. & Kushner, S.R. (1989). New method for generating deletions and gene replacements in Escherichia coli. Journal of Bacteriology, 171, 4617-4622. Hasan, N. & Szybalski, W. (1995). Construction of laclts and 0 laclqts expression plasmids and evaluation of the thermosensitive lac repressor. Gene, 163, 35-40.
Hultman, T., Stahl, S., Hornes, E. & Uhlen, M. (1989). Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Research, 17, 4937-4946. Issacs, H., Chao, D., Yanofsky, C. & Saier, M.H. (1994). d Mechanism of catabolite repression of tryptophanase synthesis n Escherichia coli. Microbiology, 140, 2126-2134. Kelley, R.L. & Yanofsky, C. (1982). trp aporepressor production is controlled by autogenous regulation and inefficient translation. Proceedings of the National Academy of Sciences, USA, 79, 3120-3124. 0 Lama, J. & Carrasco, L. (1992). The Journal of Biological
Chemistry 267, 15932-15937. Maurer, R., Meyer, B.J. & Ptashne, M. (1980). Gene regulation at the right operator (OR) of bacteriophage lambda. I. OR3 and autogenous negative control by repressor. Journal of Molecular Biology, 139, 147-161. 5 Murby, M., Samuelsson, E., Nguyen, T.N., Mignard, L., Power,
U., Binz, H., Uhlen, M. & Stahl, S. (1995). Hydrophobicity engineering to increase solubility and stability of a recombinant protein from respiratory syncytial virus. European Journal of Biochemistry, 230, 38-44. Nichols, B.P. & Yanofsky, C. (1983). Plasmids containing the trp 0 promoters of Escherichia coli and Serratia marcescens and their use in expressing cloned genes. Methods of Enzymology, 101, 156-164.
Snell, E.E. (1975). Tryptophanase: structure, catalytic activities, and mechanism of action. Advances n Enzymology and Related Areas of Molecular Biology, 42, 287-333. Stark, M.J.R. (1987). Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene. 61. 256-267. Stewart, V. & Yanofsky, C. (1985). Evidence for transcription antitermination control of tryptophanase operates expression of Escherichia coli K-12. Journal of Bacteriology, 164, 731-740. Suter-Crazzolara, C. & Unsicker, K. (1995). Improved expression of toxic proteins in E. coli. BioTechniques, 19, 202-204. Yanofsky, C. et al. (1981 ). The complete nucleotide sequence of tryptophan operate from E. coli. Nucleic Acids Research, 9, 24, 6647-6668. Yansura, D.G. & Bass, S. H. (1997). Application of the E. coli trp promoter. Methods in Molecular Biology, 62, 55-62. Yansura, D.G. & Henner, D.J. (1990). Use for Escherichia coli trp promoter for direct expression of proteins. In Anonymous, Methods of Enzymology. (pp. 54-60). San Diego, CA: Academic Press, Inc.
Claims (21)
- NOVELTY OF THE INVENTION CLAIMS • 5 1.- A method for producing a recombinant protein of interest whose gene is placed under the control of the Ptrp promoter of the tryptophan operon, characterized in that the method comprises the following steps: a) transforming a prokaryotic cell with a vector containing a nucleic acid sequence that is capable of inactivating the gene it encodes • 10 for a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell, and integrating said sequence into the DNA of said host cell, and before, subsequently or simultaneously, introducing into said prokaryotic cell all or a portion of the sequence of a promoter that is followed, in the 3 'position, by a 1d nucleic acid sequence that encodes a molecule that is ribonucleotide or protein by nature, and that acts negatively on • the Ptrp promoter or its transcription product; b) transforming said prokaryotic cell with a vector containing a gene encoding said recombinant protein of interest; C) cultivating said transformed cell in a culture medium that allows the expression of the recombinant protein; and d) recovering the recombinant protein from the culture medium or from said transformed cell.
- 2. - The method for producing a recombinant protein of interest according to claim 1, wherein the nucleic acid sequence that is capable of inactivating the gene encoding a TnaA • tryptophanase is introduced into the DNA of the host prokaryotic cell according to the chromosomal integration method described in Example 1 or 2.
- 3. A method for producing a recombinant protein of interest according to any of claims 1 or 2. 2, wherein said nucleic acid sequence introduced into said host cell is • 10 introduces without any other element of DNA, which could allow a selective utility that will be associated with it.
- 4. The method for producing a recombinant protein of interest according to one of claims 1 to 3, wherein said nucleic acid introduced into said host cell is introduced into the 1 d locus of the tryptophanase operand.
- 5.- The method to produce a recombinant protein of interest • according to one of claims 1 to 4, wherein said method also comprises, between step a) and step b), a step of resolution and selection.
- 6. The method for producing a recombinant protein of interest according to one of claims 1 to 5, wherein the induction of said promoter is followed, at the 3 'position, by a nucleic acid sequence. which encodes a molecule that is ribonucleotide or protein by nature, and which acts negatively on the Ptrp promoter or its transcription product is obtained by any means, allowing an inhibition or activation effect on said promoter to be exerted. 5 7.- The production method in accordance with the claim 6, wherein the induction of said promoter is followed by, in the 3 'position, a nucleic acid sequence that encodes a molecule that is ribonucleotide or protein in nature, and that acts negatively on the Ptrp promoter or its product.
- Transcription is obtained either by: 10 a) choosing a suitable carbon source in the culture medium or b) adding tryptophan to the culture medium; or c) by a combination of a) and b).
- 8. The method according to one of claims 1 to 7, wherein said host prokaryotic cell is also a Gram-negative 1 d bacterium.
- 9. The method according to one of claims 1 to 8, further characterized in that the host prokaryotic cell is E. coli.
- 10.- The first construction to transform a cell 20 prokaryotic host that can be transformed with a second construct to express a gene encoding a recombinant protein of interest placed under the control of the tryptophan operon Ptrp promoter in a prokaryotic host cell, wherein the first construct comprises a nucleic acid sequence that is capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell.
- 11. The first construct according to claim 10, further characterized in that it also comprises, towards the 5 'end of said nucleic acid sequence capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into the nucleic acid sequence. said host cell, all or a part of the nucleic acid sequence of the Ptna promoter of the tryptophanase operon.
- 12. The first construction according to any of claims 10 and 11, further characterized in that said nucleic acid sequence capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell comprises a mutated fragment of the sequence encoding said TnaA tryptophanase.
- 13. The first construction according to claim 12, further characterized in that said mutated fragment is obtained by inserting a stop codon in a position such that the sequence of the mutated fragment obtained in this way encodes a fragment of the missing protein. tryptophanase activity.
- 14. The first construction according to any of claims 12 and 13, further characterized in that said mutated fragment is a mutated fragment of the sequence encoding the TnaA tryptophanase of said host cell. 16.
- The first construction according to claim 10, further characterized in that said nucleic acid sequence capable of inactivating the gene encoding a TnaA tryptophanase when said nucleic acid sequence is introduced into said host cell is the nucleic acid sequence comprising all or part of the sequence of a promoter followed, at the 3 'position, by a nucleic acid sequence that encodes a molecule that is ribonucleotide or protein in nature, and that acts negatively on the Ptrp promoter or its product. transcription.
- 16. The first construct according to claim 15, further characterized in that said promoter is followed, in the 3 'position, by a nucleic acid sequence that encodes a molecule that is ribonucleotide or protein in nature, and that acts negatively on the Ptrp promoter, in all or a part that allows the activity of the promoter, of the Ptna promoter of the tryptophanase operon.
- 17. The first construction according to claim 16, further characterized in that said nucleic acid sequence that codes for a molecule that is ribonucleotide or protein by nature that acts negatively on the Ptrp promoter, is the sequence that codes for the TrpR aporrepressant of the tryptophan operon or one of its biologically active fragments.
- 18. - The vector containing a first construction according to one of claims 10 to 17.
- 19. The vector according to claim 18, further characterized in that it is the vector pMAK705 [tnaAt] as defined in example 1 or the vector pMAK70d [Ptna: trpR: 3'tna].
- 20. The prokaryotic host cell transformed with a vector according to any of claims 18 and 19.
- 21. The host prokaryotic cell according to claim 20, further characterized because it is E. coli.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR98/04638 | 1998-04-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA00010057A true MXPA00010057A (en) | 2001-09-07 |
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