Patent Application
20070015248
The present invention relates to an expression vector for the use in an auxotrophic, prokaryotic host cell and relates to an expression system containing an expression vector and an auxotrophic, prokaryotic host cell. The invention furthermore relates to an antibiotic-free fermentation medium containing an expression vector as mentioned above as well as to a method for antibiotic-free expression of peptides/proteins.
The use of expression vectors, for example plasmids, for the expression of for example therapeutic peptides/proteins has been known for a long time. Thus, by large scale production of recombinant proteins for example in E. coli a wide variety of proteins could be made available for biochemical research, biotechnology and even for medical therapy. An example of successful application of this methodology is the bacterial production of an antigen-binding immunoglobulin Fab fragment in medium and high cell densities (Carter et al., 1992; Schiweck and Skerra, 1995). In general, heterologous biosynthesis with respect to the genetechnological production of therapeutic proteins has gained increasing interest in the last decade.
A problem that often encountered in the production on an industrial scale is a reduction in the yield of recombinant protein per cell. One reason for this phenomenon is the loss of functional plasmid from cultures with high cell density due to a segregating or structural instability of genetically safe expression vectors (Corchero and Villaverde, 1998). In contrast to natural vectors bearing ColEI such plasmids lack mobility and distribution functions usually ensuring inheritance to the progeny. Under laboratory conditions, reduced genetic stability is compensated for by antibiotic selection of the bacteria using a resistance marker. It is difficult, however, to maintain this selective pressure under fermentation conditions, and a loss of functional plasmid thus can occur (Zabriskie and Arcuri, 1986; Broesamle et al., 2000).
Another important parameter for the successful production of a recombinant protein in high yield is the selection of an adequate bacterial host strain able to significantly influence the concentration of the gene product synthesized (Sambrook et al., 1989). One of these is for example the E. coli K12 strain JM83 which has been successfully used since many years on a laboratory scale for the expression of antibody fragments via periplasmatic secretion (Skerra et al., 1991; Fiedler and Skerra, 1999). The E. coli K12 strain JM83 is an example of an auxotropic strain, i.e., the strain is unable to produce an essential amino acid by itself. In the case of E. coli K12 strain JM83 the amino acid is proline. This host strain shows a superior functional expression as compared to many other E. coli strains if the protein is secreted into the bacterial periplasm (Skerra 1994 a, b). This proline-auxotropic strain, however, could not be used for fermentation experiments due to its inability to grow on minimal medium. The use of synthetic media for fermentation is generally preferred for an industrial production scale since the growth properties can be controlled in a simple manner by using a defined carbon source, in most of the cases glucose or glycerol (Yee and Blanch, 1992). In the case of JM83 it was found, however, that a supplementation of a glucose/ammonia/mineral salt medium with proline is not feasible since this amino acid is preferably metabolized both as a carbon and as a nitrogen source (Neidhard et al., 1990) and therefore dictates the growth rate.
The proBA locus (Mahan and Csonka, 1983; Deucht et al., 1984) encodes γ-glutamyl kinase (ProB) and glutamate-5-semialdehyde dehydrogenase (ProA) both playing a key role in the proline biosynthetic pathway.
In Gene 274 (2001) 111-118, M. Fiedler and A. Skerra disclose an expression vector, more particularly a plasmid, comprising the following elements operably linked to each other: a repressor for the control of expression (TetR), a promoter for expression (tet), an antibiotic resistance gene for chloramphenicol as well as proBA. The above-mentioned publication, however, does not describe that the plasmid can be used with the amino acid as the only selection means and the only marker. Furthermore, Fiedler et al. do not disclose the use of the above-mentioned plasmid for the production of recombinant proteins in an antibiotic-free fermentation medium.
The use of antibiotics for the selection of plasmids is a significant disadvantage particularly in the preparation of therapeutic proteins. An antibiotic-free process would achieve a higher acceptance by the regulatory authorities (e.g. FDA, EMEA) since the product will be safer for the patient, and because a markedly less cost-intensive process could be devised due to reduced final product analytics (depletion of the antibiotic in the product).
Therefore, it is the object of the present invention to provide a method for antibiotic-free expression of peptides/proteins. It is another object of the invention to provide a fermentation medium and an expression system which are suitable for the use in this method.
These objects are achieved by the subject matter of the independent claims. Preferred embodiments are mentioned in the dependent claims.
In the following, the present invention will be explained in more detail with respect to drawings and the accompanying Examples. It should be understood that the scope of the invention is not restricted to these embodiments which are provided for illustration purposes only.
In the Figures, the steps for the construction of pSCIL043 are shown:
All relevant gene areas were highlighted by colours. The following regions are important for the function of the vector: lacI (repressor, red); Km (antibiotic resistance, green); proBA (selectable marker, yellow); t0 (terminator, dark green); tac (promoter, blue).
The t0 terminator (94 bp) was amplified by means of PCR directly from the chromosomal Lambda DNA obtained from MBI-Fermentas.
pACYC177 was used as a template for the Km cassette. The PCR product was subcloned into pGEM Teasy (Promega) after purification by the Gel Extraction Kit (Qiagen).
To exchange the antibiotics resistances and to introduce two new restriction sites (NheI and ApaI) pSCIL001 was amplified by means of primers flanking the Amp cassette.
By restriction with ApaI/NheI the Km cassette is again cut out of the vector. By using EcoRI, EcoRV and AflIII for the restrictions the vector is only linearized. 1: 1 kb marker MBI; 2: pSCIL002 ApaI/NheI; 3: pSCIL002 EcoRI; 4: pSCIL002 EcoRV; 5: pSCIL002 AflIII.
By means of the above-mentioned primers the proBA determinant could be amplified. The fragment contains the native terminator of the proBA gene.
To confirm correct insertion of the proBA determinant in pSCIL002 the resulting plasmid was cut with EcoRV whereby the following fragments should be generated: 2479, 1542 and 999 bp. In case of a negative result for pSCIL002 the vector should only be linearized. 1: 1 kb marker MBI; 2: pSCIL003 EcoRV; 3: pSCIL003 uncut.
On complete medium all strains (wild type=XL1Blue) grow equal to the complemented strain (not shown). On mineral salt medium only the strain containing pSCIL003, but not the strain containing pSCIL002 is able to grow.
By means of the above-mentioned primers the lacI repressor could be amplified directly from K12 chromosomal DNA. The gene was ligated into the subcloning vector pGEM Teasy (Promega) and the integrity of the PCR product was confirmed by restriction analysis and sequencing.
By the PCR product one of the two EcoRI restriction sites was destroyed during ligation.
From the synthesized product the MIA gene was amplified by means of PCR. After subcloning into pGEM Teasy and restriction the fragment was separated by agarose gel electrophoresis and purified. Afterwards the DNA fragment was ligated into pSCIL008EcoRI/PstI.
The expression assay for pSCIL043 was performed with JM83 in mineral salt medium. At an OD of 0.8 the induction was performed with 1 mM IPTG. The expression prior to induction as well as during the hours following induction is shown 1: marker 2: SP83-043 (MSM) prior to induction; 3: SP83-043 (MSM) 1 h following induction; 4: SP83-043 (MSM) 2 h following induction; 5. SP83-043 (MSM) 3 h following induction
After the expression had been terminated the cells were diluted and plated on solid LB medium. The colonies grown were picked onto LB-Agar without antibiotics and with antibiotics, respectively. All colonies grew on both media resulting in a plasmid stability of 100%. In contrast, a plasmid stability of 12-35% was detected in an expression assay performed in parallel in E. coli BL21.
The key feature of the present invention is the generation of an expression system enabling an antibiotic-free fermentation, i.e. protein/peptide expression.
In particular, the present invention relates to the following:
According to a first aspect the invention relates to an expression vector for the use in an auxotrophic, prokaryotic host cell comprising the following components operably linked to each other:
Preferably, the regulatory sequence is a tac promoter having a ribosomal binding site. Instead of this promoter, however, many other promoters can be used, particularly all promoters on the basis of the lac promoter. Examples for these promoters are the pac, rac, trc, tic promoter. For further information with respect to promoters useful in the context of the present invention see Brosius J. et al. in J Biol Chem. 1985 Mar. 25; 260(6):3539-41, “Spacing of the −10 and −35 regions in the tac promoter. Effect on its in vivo activity.” and Donovan R. S. et al. in J Ind Microbiol. 1996 March; 16(3):145-54, “Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter.”
It is further possible to use the PL, PR promoters from phage Lambda and ara, the arabinose promoter, which are well-known in the field of molecular biology.
According to one embodiment the expression vector according to the invention has a terminator for the termination of transcription for which the use of the t0 terminator from bacteriophage Lambda is particularly preferred. In principle, any functional terminator can be used in this position. Numerous examples exist therefor since most operons or genes are flanked by a terminator structure to prevent read-through by the polymerase.
The expression vector according to the invention furthermore carries a repressor gene for which the lacI gene is particularly preferred. A repressor gene as used herein comprises any gene encoding a protein which prevents the transcription of genes after binding to the promoter region. An example according to the invention of these is the above-mentioned lac repressor gene (particularly the lac repressor gene from E. coli K12; see examples).
Another well-known repressor is the CI repressor from phage Lambda which, however, does not bind to the lac hybrid promoters but only to the PL and PR promoters. Furthermore, the AraC repressor can be used since it suppresses transcription from the pBAD (ara) promoter. Numerous other examples of repressors which can be employed in the same way are known to those skilled in the art.
Sequence information in this respect can be found for example in the following references
According to a preferred embodiment the expression vector according to the invention contains a ribosomal binding site having the sequence AGGAGA.
A ribosomal binding site is a binding site for the small subunit of the ribosome on the mRNA upstream of the start codon. In prokaryotes this generally corresponds to the Shine-Dalgarno (SD) sequence which is often localized three to eleven nucleotides upstream of the start codon and shows complementarity to a region at the 3′ end of the 16sRNA. The Escherichia coli SD consensus sequence is UAAGGAGGU.
According to another embodiment of the expression vector according to the invention the first selectable marker gene is an antibiotic resistance gene, preferably a kanamycin resistance gene. Antibiotic resistance genes which can be used in the present context are for example: ampicillin resistance (amp); tetracycline resistance (tet); chloramphenicol resistance (cat); neomycin resistance (corresponding to the kanamycin resistance gene; e.g. the Km resistance gene from vector pACYC177; see examples).
Information with respect to pACYC177 can be found in: Rose, R. E. The nucleotide sequence of pACYC177, Nucleic Acids Res. 16 (1), 356 (1988), as well as at the link http://www.fermentas.com/techinfo/nucleicacids/sequences/pacyc177.txt.
As described above, the expression vector according to the invention contains a second selectable marker gene wherein the marker gene encodes an amino acid not expressed by the auxotropic host. This second selectable marker gene is also referred to a auxotrophy gene herein. The second selectable marker preferably is proBA, the methionine auxotrophy gene metB and/or the leucine auxotrophy gene leuB.
The following data from the literature with respect to proBA describe the genes and their activity: Baich, A., (1969) Proline synthesis in Escherichia coli. A proline inhibitable-glutamic acid kinase. Biochim Biophys Acta 192, 462-467. Baich, A., (1971) The biosynthesis of proline in Escherichia coli, phosphate-dependent glutamate γ-semialdehyde dehydrogenase (NADP), the second enzyme in the pathway. Biochim Biophys Acta 244, 129-134.
In the following references with respect to metB the gene and its activity have been described: Duchange, N., Zakin, M. M., Ferrara, P., Saint-Girons, I., Park, I., Tran, S. V., Py, M. C. and Cohen, G. N. Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5′- and 3′-flanking regions of the metBL operon, J. Biol. Chem. 258 (24), 14868-14871 (1983).
Sequence data can be found at:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=146844&itemID=5&view=gb withparts
Data from the literature concerning leuB in which the gene has been described can be found in: Blattner, F. R., Plunkett, G. III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B. and Shao, Y. The complete genome sequence of Escherichia coli K-12, Science 277 (5331), 1453-1474 (1997).
Sequence data can be found at:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=1786250&itemID=37&view=g bwithparts
By means of the expression vector according to the invention numerous coding sequences can be expressed which can be chosen without any limitation. The expression vector according to the invention has been found particularly advantageous for the expression of the G-CSF, MIA and/or BMP coding sequences. Furthermore, all therapeutically relevant proteins can be expressed (e.g. tPA, TNF, HGF, NGF, proteases such as trypsin, thrombin, enterokinase, β-TGF, interferons, erythropoietin, insulin, Factor VII, Factor VIII, single chain antibodies, Affilin™ as well as fusions of these proteins, G protein coupled receptors as well as the domains thereof, and the pro-forms of these proteins) to be produced as inclusion bodies or soluble variations thereof. Finally, also the expression of GM-CSF, M-CSF, interleukins, interferons, calcitonin, caspases, VEGF, Factor III, Factor X, Factor Xa, Factor XII, Factor XIIa, GDF, IGF, metalloproteases, antibodies, antibody fragments or immunotoxins can be considered.
According to another embodiment the vector according to the invention is a high copy plasmid. A high copy plasmid or high copy number plasmid (also called multi copy plasmid). respectively, refers to small plasmids (usually <15 k b) present in a high copy number (>20 plasmids/chromosome). Such plasmids such as for example pUC plasmids derived from pBR322 are often employed as cloning and expression vectors.
In a specific embodiment the expression vector according to the invention contains the following components operably linked to each other:
Furthermore, the invention relates to the expression vector pSCIL008 containing the following components operably linked to each other:
According to a second aspect the present invention relates to an expression system comprising the following components:
According to the invention the host cell preferably is an auxotropic E. coli cell which is auxotrophic for the amino acid proline.
The E. coli cell preferably is chosen among the strains JM106, JM108, JM109, JM83 and TB1 or the derivatives thereof. Derivatives is intended to mean strains which were genetically manipulated but retain the auxotrophies relevant for expression and thus can be transformed by the vectors according to the invention which complement the amino acid auxotrophies. The invention also comprises those strains that were manipulated by methods according to the prior art to acquire auxotrophies which can be used as selectable markers.
The above-mentioned E. coli strains have been deposited under the following DSMZ numbers:
JM83 DSMZ no. 3947
JM108 DSMZ no. 5585
JM109 DSMZ no. 3423
A detailed description of JM106 can be found in Yanisch-Perron et al., 1985. Information for TB-1 is available in Biochemistry 23:3663-3667 1984.
According to a third aspect the invention relates to an antibiotic-free fermentation medium comprising the following components:
According to a forth aspect the present invention relates to a method for antibiotic-free expression of peptides/proteins comprising the die following steps:
According to one embodiment the selection in step b) is additionally carried out by an antibiotic.
In other words, in the present invention particularly the fermentation process, i.e. the actual process stage for the expression of the peptides/proteins, is performed in a completely antibiotic-free environment. In this manner, the problems mentioned in the beginning which accompany the use of antibiotics for the selection of plasmids such as for example concerns of the regulatory authorities, product safety, final product analytics (depletion of the antibiotic in the product) and the risks and costs associated therewith can be circumvented. By the approach according to the invention the selective pressure during the fermentation process is still maintained, and this without using antibiotics in the fermentation medium.
1) Origin, Phenotype and Genotype of the Expression Strain
2) Expression of MIA
3) References
MIA gene sequence (synthetic gene):
Detailed Description of the pSCIL043 Expression Vector
In the following, a detailed description of plasmid pSCIL043 is given. The MunI/EcoRI site (G1AATTG) which is no longer intact due to the cloning strategy is counted as the first base. A plasmid map is shown in
Preparation of the Production Plasmid pSCIL043
1. Insertion of a Transcription Terminators into the Starting Vector=pSCIL001
1.1. Restriction of the Starting Vector
Amplification of the t0 terminator by means of PCR using the following primers (
Exchange of the Antibiotic Resistance in pSCIL001=pSCIL002
1.2. Amplification of the Kanamycin Cassette
Amplification of the Km cassette (990 bp) from pACYC177 (NEB) by means of PCR using the following primers (
Amplification of vector pSCIL001 (1462 bp) without Amp cassette (i.e. the primers used flanked the Amp cassette present in pUC19) by means of PCR using the following primers (
Amplification of the proBA operon (2520 bp) by means of PCR was done using the following primers (
3.2. Cloning of the proBA Determinant into pSCIL002ApaI and Functional Assay
Amplification of the lacI gene including the native promoter (1160 bp) by PCR was performed using the following primers (
4.2. Introduction of the Repressor Gene into pSCIL003=pSCIL004a
For the amplification of the tac promoter (67 bp) by means of PCR the following primers were used. Vector pKK233-3 (Amersham, see Annex) was used as a template for the PCR.
An expression vector according to the invention is transformed into competent E. coli JM83 and E. coli BL21 cells using methods corresponding to the prior art. These cells are first plated on LB agar and incubated at 37° C. Afterwards they must be adapted to mineral salt medium. For this purpose a clone is transferred from the LB agar plate onto a plate containing mineral salt medium agar and is incubated at 37° C. To improve the adaptation to this medium a clone from the agar plate containing mineral salt medium is transferred to another agar plate with mineral salt medium and is incubated at 37° C. 100 ml of mineral salt medium are inoculated with a clone from this plate. The incubation is performed at 180 rpm at 37° C. overnight up to an optical density of OD600=3. At this OD glycerol cultures are prepared from the liquid culture which are composed of 800 μl of cell suspension and 200 μl glycerol.
In this Example two fermentations are described in 10 1 laboratory fermenters (B. Braun Biotech). The first is processed with E. coli JM83. The second is performed with E. coli BL21 in which the selectable marker is inactive since BL21 is not auxotrophic. The fermentations are performed as fed batch processes. The batch volume is 6 1. 2 1 of substrate (feed) are dosed to obtain a final volume of 8 1.
As inoculate one glycerol culture in 100 ml of mineral salt medium is incubated overnight at 180 rpm and 37° C. (1st pre-culture). Four flasks with 100 ml of mineral salt medium are inoculated with a partial volume of the first pre-culture and incubated at 180 rpm and 37° C. (2nd pre-culture). At an optical density of e.g. OD600=3 the cell suspension is centrifuged and the cell pellet is resuspended in 50 ml of physiological saline.
The batch phase starts with inoculation and ends at a defined optical density, e.g. OD600=18, with the start of the fed batch phase, i.e. with the start of substrate dosing. Dosing of the substrate (feed flow) is performed according to an exponential function of the form feed flow=const*exp(μ1*t) wherein μ is the specific growth rate and wherein μ1=const<μmax. I.e. the amount of substrate at each point of time is always lower than the maximum demand of the cells at this time thereby achieving a submaximal growth rate. For example, μ1=0.35 h−1 is chosen.
At a defined optical density, e.g. OD600=75, the protein expression is induced by addition of IPTG, e.g. 1 mM. At this time another specific growth rate μ2 is adjusted for the cells, e.g. μ2=0.1 h−1, by means of substrate dosing (feed flow) The process is terminated after a defined incubation period, e.g. 4 h.
The oxygen demand which continues to increase with increasing cell density is kept constant at e.g. 20% saturation using a cascade control for the oxygen partial pressure pO2. With increasing demand this results in an increase in agitator rotational speed. If a maximum rotational speed is obtained the gassing rate with air is increased. If the gassing rate reaches its maximum, pure oxygen is dosed with the incoming air.
Nitrogen is supplied via pH regulation wherein ammonia is used as base. Phosphoric acid serves as acid. An anti-foaming agent is automatically dosed in the case of strong foam formation.
Samples for the determination of plasmid stability are retrieved at different time points of fermentation. While a total loss of plasmid and thereby of protein expression is very rapidly encountered in BL21, 100% plasmid stability can be detected in JM83 during the whole fermentation process. In parallel to these samples the plasmids are isolated from several samples (in hourly intervals in the course of the fermentation). The plasmid integrity in JM83 is determined by means of different endonucleases and is found to be unaffected over the whole fermentation process. The restriction patterns showed the same bands as in the starting plasmid.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10360483.9 | Dec 2003 | DE | national |
This application is a continuation of PCT International Patent Application No. PCT/EP2004/014635, filed Dec. 22, 2004, which claims priority to German Patent Application No. 10360483.9, filed Dec. 22, 2003, the disclosures of each of which are incorporated herein by reference in their entitety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP04/14635 | Dec 2004 | US |
| Child | 11471799 | Jun 2006 | US |
