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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

• This invention relates to the production of recombinant proteins and in particular the use of a promoter suitable for the expression of foreign proteins in bacteria, particularly Escherichia coli.
• E. coli The large scale production of foreign proteins in E. coli requires the use of a strong promoter that is well regulated, allowing the growth phase and the induction phase to be separated. Without this regulation a highly expressed gene can place a constraint on cell growth and plasmid stability, even if its product is not actually toxic.
• E. coli or coliphage promoters only a few satisfy these requirements: e.g. p L from lambda, lac and trp from E. coli, the hybrid trp-lac (tac) promoter and the T7 RN ⁇ polymerase promoter. All of these rely on either a temperature shift or the addition of a chemical to induce their activity. A temperature shift may cause the recombinant protein to form inclusion bodies, and furthermore by activating the heat-shock response may lead to increased proteolysis. Chemical agents are often expensive and require subsequent removal from the recombinant protein at the purification stage.
• nirB promoter has been isolated from E. coli. where it directs expression of an operon which includes the nitrite reductase gene nirB (1), and nirD, nirC and cysG (2). It is regulated both by nitrite and by changes in the oxygen tension of the environment, becoming active when deprived of oxygen (3). Response to anaerobiosis is mediated through the protein FNR, acting as a transcriptional activator, in a mechanism common to many anaerobic respiratory genes (1).
• nifA promoter of Rhizobium meliloti (7) and the Vitreoscilla sp. haemoglobin (VHb) promoter (8) are examples of promoters with such a microaerobic optimum. Indeed these promote relatively little transcription under fully anaerobic conditions.
• WO 89/03883 relates to the use of changes in the level of oxygen available in the culture medium to control the expression of foreign DNA in a host cell.
• this specification is concerned with the use of the VHb promoter referred to above.
• This promoter requires the presence of small amounts of oxygen for optimum expression makes expression difficult to regulate since it is difficult to control the amount of oxygen in the culture with sufficient precision.
• the present invention provides a process for the expression of a heterologous protein, which process comprises maintaining under anaerobic conditions bacteria in which expression of the said protein is under the control of a promoter whose activity is induced by anaerobic conditions. Expression of the protein can thus occur in a bacterial culture under anaerobic conditions.
• VHb promoter induced by anaerobic conditions has considerable advantages over promoters such as the VHb promoter in terms of the ease with which such conditions can be maintained. Thus, particularly in the case of large scale fermentation involving high cell densities, it is much easier to maintain completely anaerobic conditions rather than the microaerobic conditions required by the VHb promoter.
• DNA encoding the heterologouis protein of interest will be placed under control of the promoter, for example in a replicable expression sector such as a plasmid vector.
• Bacteria for example an appropriate strain of E. Coli. transformed with the expression vector are then used for expression of the heterologous protein.
• the process involves a growth phase in which the bacteria are allowed to grow whilst a high oxygen tension is maintained in order to prevent premature activation of the promoter.
• an induction phase in which the bacteria are maintained under anaerobic conditions in order to activate the promoter.
• the aerobic conditions required during the growth phase can be maintained, for example, by sparging with an oxygen containing gas such as pure oxygen, oxygen enriched air or air.
• the anaerobic conditions required to activate the promoter can be established and maintained by sparging with an inert gas such as nitrogen.
• the anaerobic conditions may be maintained for up to 19 hours, for example for from 5 to 19 hours.
• the process according to the invention can be applied to the production of any heterologous protein that can be produced in bacteria.
• the DNA encoding the protein can be incorporated into an expression vector under control of the promoter by use of standard recombinant DNA techniques.
• the expression vector will also generally include other elements required for efficient expression.
• the heterologous protein can be recovered and purified by conventional methods.
• the heterologous protein typically is a physiologically active polypeptide such as an enzyme.
• the polypeptide may be a polypeptide drug.
• Polypeptide drugs which may be expressed by the present process therefore include tissue plasminogen activator, luteinizing hormone releasing hormone, human growth hormone, insulin, erythropoietin, an interferon such as ⁇ -interferon and calcitonin.
• the heterologous protein may be a polypeptide immunogen capable of inducing an immune reponse in a human or animal.
• a polypeptide immunogen may comprise an antigenic determinant of a pathogenic organism.
• the polypeptide immunogen therefore typically comprises an antigenic sequence derived from a virus, bacterium, fungus, yeast or parasite.
• the immunogen may be an antigenic sequence derived from a type of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, hepatitis A or B virus, herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus, influenza virus, coxsackie virus, the cell surface antigen CD4, Chlamydia trachomatis and Plasmodium falciparum.
• HIV human immunodeficiency virus
• the immunogen may comprise the CD4 receptor binding site from HIV, for example from HIV-1 or -2.
• Other useful immunogens include E. coli heat labile toxin B subunit (LT-B), E. coli K88 antigens, P.69 protein from B. pertussis, tetanus toxin fragment C and antigens of flukes, mycoplasma, roundworms, tapeworms, rabies virus and rotavirus.
• the immunogen may be a tumour-specific antigen.
• nirB promoter can be activated by the use of anaerobic conditions and does not show a microaerobic optimum.
• the nirB promoter also shows a strength and an induction ratio
• nirB promoter (the ratio of expression levels before and after induction) which are comparable to known bacterial promoters such as the tac promoter. For this reason the nirB promoter is particularly suitable for use according to the invention.
• the nirB promoter has been isolated from E. coli and is particularly suitable for the control of expression in E. coli.
• nirB promoter is regulated both by nitrite and by changes in oxygen tension and the promoter can be used according to the invetion in this form. However, it is preferred to use only that part of the nirB promoter which responds solely to anaerobiosis.
• references to the nirB promoter refer to the promoter itself or a part or derivative thereof which is capable of promoting expression of a coding sequence under anaerobic conditions.
• proteins which have been expressed in E. coli under control of a form of the nirB promoter which lacks the nitrite response regions are tetanus toxin fragment C and Bordetella pertussis pertactin. These proteins are both potential components of sub-unit vaccines against tetanus and whooping cough respectively.
• the present invention also proivdes a DNA molecule comprising the nirB promoter sequence operably linked to a DNA sequence encoding a protein heterologous to the host bacteria.
• the invention further provides a replicable expression vector, suitable for use in bacteria, in which a DNA sequence encoding a heterologous protein is under the control of a promoter whose activity is induced by anaerobic conditions.
• a preferred vector comprises the nirB promoter sequence operably linked to a DNA sequence encoding a protein heterologous to the host bacteria.
• the invention also provides bacteria, particularly an E. coli strain, transformed with such an expression vector. A bacterial culture containing the expression vector can thus be provided.
• the invention also provides the use of a promoter activated by anaerobic conditions for the control of expression of a heterologous protein in bacteria. More particularly the invention provides the use of the nirB promoter for the control of expression of a heterologous protein in bacteria, preferably an E. coli strain.
• Figure 1 shows a Coomassie blue stained sodium dodecyl sulphate (SDS)-polyacrylamide gel of E. coli extracts containing pTETnir215 or pTETnir36; and
• Figure 2 shows the accumulation of tetanus toxin fragment C, as % total cell protein (top), as a function of time. Dissolved oxygen tension (DOT) is also shown as % saturation.
• DOT Dissolved oxygen tension
• E. coli strain MM294 9 and plasmids pTETnir15, pPERnir36, pTETnir215 and, for purposes of comparison pTETtac315.
• Expression plasmids pTETnir15 and pPERnir36 were constructed from pTETtacll5 (10) and pPERtac36 (11) by replacing in each the EcoRI-ApaI region (1354/bp and 1294/bp respectively) containing the lacI gene and tac promoter with the pair of oligos 1 and 2 which contain the nirB promoter:
• Oligo-1 5'-AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAAT
• Oligo-2 3'-GTCCATTTAAACTACATGTAGTTTACCATGGGGAACGACTTA
• Plasmid pTETnir215 was constructed by ligating the 1530 bp AatII-BamHI fragment of pTETnir15, containing the nirB promoter and fragment C gene, with the 1983 bp BamHI-AatII fragment from pTETlac5 (13), containing the pUC-based origin of replication and ⁇ - lactamase gene.
• the plasmid pTETtac315 contains the tetanus toxin fragment C sequence under control of the tac promoter (13).
• the plasmid pTETnir15 contains the gene for tetanus toxin fragment C under nirB promoter control.
• the version of the nirB promoter used was based on the F2 DR25X variant described by Bell and co-workers (6), the BamHI (GGATCC) site of which was replaced by a Kpnl (GGTACC) site, involving only a minimal sequence change.
• An equivalent plasmid, pPERnir36 had the gene for B. pertussis pertactin [also referred to as P69 (11)] in place of the fragment C gene.
• fragment C is mRNA-limited, over a wide range of mRNA levels, and consequently a good indicator of promoter strength (13).
• pertactin becomes mRNA-saturated at relatively low concentrations. Fragment C synthesis was therefore chosen as a model system for studying promoter strength.
• Fragment C levels were increased about two-fold when the pAT153 replicon of pTETnir15 was replaced by the higher copy-number pUC19 replicon, a similar improvement to that observed in lac (unpublished observations) and tac expression vectors (10,13).
• the improved vector, pTETnir215, was used for later experiments as the higher fragment C levels could be more accurately estimated.
• the polarographic probe for measuring DOT was zero-calibrated by sparging the fermenter with oxygen free nitrogen.
• 1.5 1 of L-Broth containing 10 mgml -1 glucose and 100 ⁇ gml -1 ampicillin was inoculated with 45 ml of overnight culture of E. coli containing either pTETnir215 or pPERnir36. This was grown aerobically for a period of 4-5h at pH7.2, stirrer rate of 750rpm and air input at up to 1.3vvm to maintain a DOT value in excess of 75% of saturation after which time the A 650 was typically 1.5-3.0.
• Inductions were carried out on cultures of E.coli containing pTETnir215 or pPERnir36. Analysis of samples showed that recombinant protein accumulated following anaerobiosis, demonstrating that the nirB promoter is active under completely anaerobic conditions.
• Figure 1 shows Coomassie blue stained SDS-polyacrylamide gel of E. coli extracts containing pTETnir215 or pPERnir36. Extracts from pTETnir215: preinduction (lane 1) and 19 hours post-induction (lane 2); from pPERnir36: pre-induction (lane 3), and 20 hours post-induction (lane 4). Molecular weight markers are in kDa.
• Fig. 2 shows accumulation of fragment C as a function of time in an experiment as described above.
• the fragment C levels produced under these conditions were very high, approximately 20% total cell protein (tcp), comparable to those obtained by pTETtac315 which has a shortened form of the tac promoter.
• tcp total cell protein
• some bacterial oxygen-regulated promoters such as the Vitreoscilla haemoglobin promoter, have a microaerobic optimum. Inspection of the Vitreoscilla promoter sequence (8), upstream of the -10 region, revealed a run of nucleotides in which 15 out of 22 matched the consensus FNR-binding site (4). Furthermore, the distance between this sequence and the -10 region is consistent with the critical distance from the putative FNR-binding site to the -10 homology sequence in the nirB promoter (6). This finding might well lead to the assumption that the nirB promoter shows a microaerobic optimum.
• nirB promoter sequences in pTETnir15, F2 DR25X (6), the FNR consensus sequence (4), wild-type nirB promoter sequence (1) and the Vitreoscilla haemoglobin (Hb) promoter sequence (8) is shown below. Restriction enzyme sites and -10 homology regions and Shine-Dalgarno sequences (SD) are underlined. conserveed nucleotides of the FNR consensus sequence are shown in bold italics. EcoRI FNR KpnI
• the background fragment C content of a pre-induction sample on a Western blot was investigated using the method described previously (15).
• the uninduced level resulting from a tac-driven plasmid, pTETtac315, which expresses the fragment C gene at a similar level when induced (13) was also investigated.
• the induction ratio of the nirB promoter closely matches that of the tightly regulated tac promoter.
• nirB promoter is able to express very efficiently genes for both tetanus toxin fragment C and B. pertussis pertactin.
• the nirB promoter produced similar levels of fragment C to a shortened version of the tac promoter. It has previously been shown that this version produces approximately 50% of the steady-state mRNA level of the full-length tac promoter (10,13). Since it has been possible to use fragment C synthesis to discriminate between promoters with activities up to that of the full-length tac promoter (13), it is likely that under the conditions described in the above Examples the nirB promoter has approximately half the activity of the full tac promoter.
• nirB its induction ratio was investigated by measuring fragment C levels before and after induction. The ratio was found to be very similar to the value obtained for the tac promoter. These values would be expected to approximate to the corresponding mRNA ratios because, over most of this range, fragment C levels are directly proportional to mRNA levels (13).
• the regulation of the nirB promoter expression of the B. pertussis pertactin gene was examined, which would be expected to be particularly sensitive to a poorly regulated promoter.
• the growth rate of a culture containing pPERnir36 was comparable to growth rates obtained using pTETnir215. After induction, production of pertactin resulted in levels similar to those obtained using the tac promoter (11). Both these observations are consistent with good regulation of nirB promoter activity.
• nirB inductions are carried out under different physiological conditions from those used in more conventional expression systems, it is possible that the nature of the product might be affected. Protein solubility in particular can sometimes be sensitive to physiological changes such as elevated temperature. It is of interest that expression of the fragment C gene under anaerobic conditions still results in a product which is fully soluble. Another possible consequence of the use of anaerobic conditions during induction might be a difference in the repertoire of proteases active in vivo. Similar patterns of lower molecular weight bands on Western blots of both C fragment and pertactin, produced under aerobic or anaerobic conditions using the nirB promoter were observed which suggests a similar range of proteolytic activity.
• VHb promoter One other oxygen-regulated promoter that has been used for heterologous expression in E. coli is the VHb promoter referred to above (16 and WO 89/03883). This is optimally active under microaerobic conditions. In contrast, no evidence has been found for the nirB promoter requiring low levels of oxygen for maximum activity; its optimum appears to be genuinely anaerobic. This presumably relates to the fact that whereas E. coli is a facultative anaerobe, Vitreoscilla sp. is an obligate aerobe. However both promoters appear to have very similar FNR-binding sites, suggesting a common mechanism at least involving FNR. It is likely that some other feature of the Vitreoscilla promoter prevents it from functioning optimally under anaerobic conditions.

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