PROMOTER, EXPRESSION VECTOR AND POLYPEPTIDE SYNTHESIS

Abstract: The invention provides a DNA promoter sequence, an expression vector comprising a promoter-gene combination in which the promoter is activated by carbon nutrient limitation, a host containing the vector and a method of expressing a polypeptide comprising cultivating the host under suitable conditions.

Description: Promoter, Expression Vector and Poiypeptide Synthesis The invention relates to a new DNA promoter sequence, an expression vector in which the promoter-gene combination comprises the new DNA promoter sequence, an expression vector comprising a promoter- ene combination in which the promoter is activated by carbon-nutrient limitation, a host containing at least one of the before-mentioned promoter-gene combinations, and a method of expressing a poiypeptide comprising cultivating the host under suitable conditions. The construction of promoter-gene expression vectors, espe¬ cially heterologous promoter-gene expression vectors, their inser¬ tion into suitable hosts, and the expression of polypeptides are now standard technology. in order to maximise the production of polypeptides it is desired to use an efficient promoter in combination with quick transcription of the appropriate gene, efficient translation of the mRNA and in many cases, efficient post-translational processing and compartmentalization of the nascent poiypeptide. In general complex media are used to obtain rapid growth of the host organism prior to expressing the gene. The success of expressing genes, especially heterologous genes, is, however, limited by a number of factors, including the efficiency of the promoter and its control. In this specification the term promoter is used for the binding site on the DNA for NA polymerase which transcribes the DNA into mRNA. A possibility to enhance the production of a poiypeptide in the case of a low efficient promoter which is used in the promoter-gene combination may, for instance, be insertion of the gene into a high copy number plasmid. This is usually possible, but may result in instability of the gene or the plasmid. Efficient transcription of a cloned insert requires the presence of a promoter which is recognised by the RNA polymerase of the host. Efficient translation requires that the mRNA bears a ribosome binding site. Many mRNA ribosome binding sites are well known in the art. They usually comprise the translational start codon (AUG or GUG) and a sequence that is complementary to the bases of the 3' end of 16s ribosomal RNA. The identified ribosomal binding sites for, for instance, E. coli vary in length from 3-9 base pairs and precede the translational codon by 3-12 bases. To initiate the expression of proteins inserted by means of a vector into a host-organism, promoters may be used which are activated by heat-shock methods or an additional control may be build in into the DNA, which gives the possibility to control the gene by addition of certain chemical compounds, e.g. addition of an inducer such as IPTG. Another possibility to control the expression of the foreign gene is the use of a promoter which is activated in the absence of certain, very specific, compounds (metabolites), e.g. a promoter which controls the expression of the genes involved in tryptophan biosynthesis which is activated in the absence of tryptophan. Besides the controllable promoters there are the constitutive promoters, i.e. promoters which are active continuous- y- i is highly desirable to develop new, highly efficient promoters, and to be able to control the promoters in the promot¬ er-gene vector after transformation of the host by means of an easy, preferably reversible procedure. As a result of extensive experimentation a new DNA promoter sequence has been found which has a very high activity and which may be activated by limitation of the carbon supply to the cultiva¬ tion medium of a host into which a vector has been inserted com¬ prising the new DNA promoter sequence. The DNA sequence of this new promoter is present in the DNA sequence as given in Table 1. The present invention therefore, relates to a DNA promoter sequence which is present in the DNA sequence as given in Table 1. The DNA sequence as given in Table 1 has been isolated from a mutated microorganism and comprises in addition to the promoter sequence also an amount of DNA which is not part of the DNA promot¬ er sequence. It will be appreciated that these additional parts of DNA, which parts also comprise DNA coding for a ribosome binding site as discussed above, may be used for other purposes, for instance as cutting places for restriction enzymes. It will further be appreciated that nucleotides not forming part of the claimed DNA promoter sequence may be removed from the sequence as given in Table 1, and/or replaced by other nucleotides. Whether or not any nucleotide is essential for the promoter sequence can be estab¬ lished very easily, by making an amended sequence by methods now very well known in the art and testing the efficiency of the thus amended promoter, e.g. by constructing deletions, or by locating the transcription start using S. -mapping. The invention especially relates to the DNA promoter sequence as given in Table 1, in which the DNA promoter sequence is present between base number 9 and base number 355, preferably between base number 140 and base number 355. More preferably the DNA promoter sequence is present between base number 200 and number 355, still more preferably between base number 260 and number 355. The above described DNA sequence (see Table 1) was isolated from an Agrobacterium radiobacter strain. It has been described that A. radiobacter strains may be grown under carbon limitation, resulting in the formation of mutant strains having an enhanced efficiency in the production of a heteropolysaccharide when grown under normal conditions. It appeared that certain mutant strains ma y produce a glucose-binding protein (GBP1) as the major cell protein during growth in continuous culture using glucose or galactose as the carbon source (J. Gen. Microb. 134 (1988) 3099-3110 and 3111-3122). An example of such a mutant strain has been described in European patent application EP-A-341775 (A. radiobacter NCIB 40018). Further, a general process for producing and isolating strains in which a carbon-containing substrate uptake system is at least partially depressed by growing the parent strain under substrate limitation has been described. It was surprisingly found that in the case of the above mentioned strain the effect of nutrient limitation was increased production of the homologous binding protein during growth on any carbon source under carbon-limited conditions. Isolation of the promoter DNA-sequence as described above from the deposited strain resulted in a promoter which is activated during growth on any carbon source under carbon-limited conditions. The use of such a promoter has the advantage that relatively simple growth media (minimal media or defined media) may be used for the growth of the host as well as during protein synthesis. In view of the above it will be appreciated that it is a feature of the invention that a promoter which is activated by carbon nutrient limitation, in A. radiobacter or any other micro¬ organism, may be used to drive the synthesis of proteins, homologous proteins as well as heterologous proteins, by combina¬ tion with the appropriate sequences in an expression vector. The proteins are expressed by cultivating a host containing the novel vector under carbon-limited conditions. In order to obtain the promoters the same methods may be used as described in this patent specification for the isolation of the DNA sequence as described in Table 1. The promoter-gene combination is preferably a heterologous promoter-gene combination. To activate the promoter carbon limitation is used, especially catabolic carbon compound limitation, preferably carbohydrate limitation, especially mono or disaccharide limitation. Especially suitable nutrient limitation compounds are glucose, sucrose, fructose, xylose, galactose and glycerol. The expression vector as described above preferably addition¬ ally comprises a secretion signal, especially a secretion signal which is homologous with the promoter. The promoter is preferably derived from DNA which controls the expression of a periplasmic carbon substrate binding enzyme. In view of the above it will be clear that the nutrient which is limited during the growth of a host comprising a vector contain¬ ing a promoter which is derived from a specific binding protein does not necessarily has to be the nutrient which corresponds with the binding protein. It will be appreciated that the nutrient compound which is used for activating the promoter should not be replaced by another compound of the same kind, as otherwise no sufficient limitation occurs. It will further be appreciated that instead of one nutrient compound also, a mixture of nutrient compounds may be used, the addition of said mixture to the fermen¬ tation broth being diminished to activate the promoter. The promoter sequences to be used in the present invention are preferably promoter sequences related to binding proteins and are derived from mutant strains of microorganisms, the mutation having occurred after cultivation of the microorganism under carbon-limit¬ ed conditions. It is to be expected that promoter sequences as indicated above derived from mutant strains obtained by growing under carbon limitation will be more efficient than the promoter sequences of the original microorganism, as the mutant strain was adapted to the specific circumstances. Microorganisms which are especially suitable . ,r the isolation of promoters to be used in the vectors of the present invention are microorganisms of the genus Agrobacterium, preferably Agrobacterium radiobacter or Agrobacterium tumefaciens, more preferably Agrobacterium radiobacter, still more preferably Agrobacterium radiobacter deposited as NCIB 40018. Although the DNA sequence given in Table 1 is a DNA sequence derived from A. radiobacter, it will be clear for the man skilled in the art that species of the same or other genera may also be modified by growth under conditions of carbon or other nutrient limitation, to give promoters which can actively drive the expres¬ sion of homologous or heterologous genes and, if necessary or desired, secretion of the expressed proteins. In any given case, simple experiments will reveal whether substrate-limited growth activates the production of a substrate-binding or other protein. Using the same techniques as described above for the isolation of the GBP1 promoter it appeared to be possible to isolate a lactose binding protein promoter, which could be succesfully inserted in A. tuπtefaciβτis (NCIB 40325) and in E. coli, thus proving the general validity of the above described concept. The DNA promoter sequence as described in Table 2 forms also part of the invention, especially between base numbers 1 and 122. For the purpose of expressing the poiypeptide, the micro-or¬ ganism which is the host for the expression vector may be grown in an aqueous nutrient medium by aerobic cultivation. The procedure may be suitable carried out as a batch process, a fed-batch process with or without fill and draw, or as a continuous process. The growth medium, with suitable control of the appropriate nutrient, can be of a relatively simple composition. A promoter in a vector of the invention is easily fully activated by control of the appropriate nutrient. No inducer is required. The poiypeptide production is especially carried out in such a way that the polypeptides are released, preferably to the periplasm in the case of a Gram-negative micro-organism, optionally followed by an osmotic shock, thus obtaining the polypeptides in a relatively pure form. The expression vectors according to the invention especially contain the promoter sequences as claimed in any one or more of claims 1 to 4. A vector according to the invention may be produced by known technology. Thus, for example, a plasmid may be constructed com¬ prising the promoter and a heterologous gene, and the combination may then be inserted into a suitable host, e.g. the microorganism from which the promoter has been derived by nutrient-limited growth. In view of the high efficiency of the promotors according to the present invetion, a suitable plasmid may be a low copy number plasmid (stringent plasmids). The vector of the invention may be introduced into the host using known technology, Thus, the vector may be introduced for example by transformation or electroporation. The gene in the vector according to the invention may very suitably be a heterologous gene for any desired poiypeptide, e.g. a detergent enzyme, a pharmaceutical protein or a food enzyme. To the extent that the vector includes DNA homologous with the promoter, this may be desirable (e.g. coding for a secretion signal) or less desirable. An undesirable gene may be excised or inactivated from the host chromosome, e.g. by point mutation. If the host is the wild type organism, production of unwanted protein may be reduced. Suitable host-organisms to be used for the insertion of the vector of the present invention may be each host which is in principle suitable for insertion of the vector. Suitable cells of higher organisms may be used, but especially micro-organisms are used, preferably bacteria, more preferably an Agrobacterium strain or an Escherichia strain, still more preferably Agrobacterium radiobacter, Agrobacterium tumefaciens or Escherichia coli. It will be appreciated that in view of the close relationship between the Agrobacterium species, as described in Bergey's Manual of Systemat¬ ic Bacteriology, Vol I, pp. 244-254, Agrobacterium is especially suitable for the promoter as claimed in any one of claims 1 to 3. Methods. Bacterial strains. A. radiobacter NCIB 40018 was obtained by growing k__ radiobacter NCIB 11883 in continuous culture under glucose limita¬ tion. A. tumefaciens (strain T37, deposited as NCIB 40325) was obtained from J. Draper, Department of Botany, University of Leicester. Escherichia coli strains JM105 (Yanisch-Perron et al, 1985) and S 1592 were used as hosts for routine cloning experi¬ ments. Media and growth conditions E. coli strains were grown at 37 C either in DYT complex medium or M9 minimal medium (Maniatis et al. , 1982) . The latter was supplemented with glucose or glycerol (5gl " ) and thiamine (0.5 mg I "1 ). Agrobacterium strains were grown in carbon-limited continuous culture at 30 C (pH 7.0, D - 0.45h ) in a defined medium (Linton et al. , 1987) containing 1.9 gl carbon source and 3 gl " ammonium sulphate. The same medium was used for growth in shake flasks except that the concentration of KH„P0, was increased from 0.75 to -1 2 4 3g 1 to provide additional buffering capacity. Growth of E. coli and A. radiobacter in carbon-limited, fed-batch culture. E. coli was grown at 37 C under fed-batch conditions in a 3 1 Biotec fermenter containing 3 1 of Medium 1. This medium consisted of 5 separate parts which were combined after sterilisation to give a complete medium of the following composition (final concentra¬ tions of individual components are given in gl ). Part 1: NaH„P0, , 1.57: NH 4 C1, 5.3; KC1, 0.745 and Na 2 S04, 0.64. Part 2: MgCl 2 , 0.12; CaCl , 0.0022; nitrilotriacetic acid (sodium salt), 0.142 and -i 5mll trace element stock-solution (see below). Part 3: glucose, 2.5. Part 4: FeS0 4 .7H 0 (filter-sterilised), 0.028. Part 5: thiamine, 0.001. The trace element stock solution contained the following (gl "1 ): concentrated HC1, 10ml; ZnO, 0.408; FeCl 3 .6H20, 5.4; MnCl 2 .4H 2 0, 2.0; CoCl 2 , 0.17; CaCl 2 .6H 2 0, 0.47, H. j B04, 0.062 and Na 2 Mo0 4 .2H 2 0, 0.124. A. radiobacter strains were grown under fed-batch conditions at 30 C in 3 1 Biotec fermenters containing 3 1 Medium 2, which was made in 4 parts as follows (final concentrations of individual components in the complete medium are given in gl ). Part 1: KH P0 4 , 0.956; K 2 HP0 4 , 2.86 and (NH^SO^ 8.0. Part 2: MgSO^I^O, 0.6; nitrilotriacetic acid (sodium salt), 0,06 and 2.0 mil trace elements solution (see above). Part 3: glucose, 2.5. Part 4: FeS0 4 .7H 2 0, 0.025. The fermenter inoculum was grown in 200 ml Medium 3 which con¬ tained (gl "1 ): Na 2 HP0 4 , 3.0; H 2 P0 4 , 3.0; (NH 4 )2S0 4> 3.0; MgS04.7H20, 0.2; CaCl 2 .2H 0,0.0147 and FeCl 3> 0.0167. Each litre of medium was supplemented with 2.0ml of a trace elements stock solution which contained (gl " ): CaCl 2 .2H 2 0, 0.66; ZnSCK, 0.18; CuS0 4 .7H 2 0, 0.16; MnS0 .4H 2 0, 0.15; CoCl 2 .6H 2 0, 0.18; H 3 B0 3> 0.1 and Na 2 Mo0 4 2H 2 0, 0.3. Both organisms were allowed to grow exponentially in the fermenters until the glucose was exhausted at which point a glucose feed was started. This contained 444 gl " glucose and 20 gl " MgS0 4 . Characterisation of periplasmic, sugar-binding proteins. Periplasmic proteins were released from cells using osmotic shock and purified using fast protein liquid chromatography (FPLC) as described previously (Cornish et al. , 1988). Binding constants were measured using equilibrium dialysis (Cornish et al. , 1988) . Antibodies to pure proteins were raised in New Zealand white rabbits. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Discontinuous SDS-PAGE was carried out with 12.5% (w/v) polyacrylamide slab gels. Western blotting was performed according to the method of Towbin et al. (1979). Assay of chloramphenicol acetyl transferase (CAT) . Cells were washed once with 30 mM Tris-HCl, pH 8.0, resuspended in the same buffer to a density of approximately 10 mg dry wt ml and broken by two passages through a French press. The activity in broken cell extracts was measured using the method of Shaw (1975). Isolation of chromosomal DNA Chromosomal DNA was isolated from strains of A. radiobacter using the method of Chow et al. (1977) . DNA was extracted from cultures which had been grown overnight at 30 C in 100 ml minimal medium (Linton et al, 1987) containing 3 gl ammonium sulphate and 2 gl glucose. Isolation of plasmids from E. coli strains. E. coli strains harbouring plasmids were grown overnight at 37°C in DYT medium containing the appropriate antibiotics. For restriction endonuclease mapping of DNA inserts, ρUC18-based plasmids were isolated from 2ml cultures using a small-scale alkaline lysis method (Maniatis et al. , 1982) . Larger amounts of high quality plasmid DNA (0.5 - l.Omg) were isolated from 50ml cultures using the modified alkaline lysis method of Birnboim and Doly (1979) described by Maniatis et al. (1982) except that the caesium chloride centrifugation step was omitted. Instead, the DNA pellet obtained after precipitation with propan-2-ol was dried under vacuum, redissolved in 2 ml TE buffer, mixed with 2.5 ml 4.4 M LiCl and allowed to stand on ice for 1 h. The precipitate that formed was removed using centrifugation and plasmid DNA was subse¬ quently precipitated by adding 10 ml ethanol to the supernatant. After centrifugation, the DNA pellet was washed with ethanol, dried under vacuum, redissolved in 0.4 ml TE buffer and incubated at 37 C for 15 min with 100 mg RNase. The DNA was then extracted with phenol (x2) followed by phenol/ chloroform/iso-amylalcohol, ethanol-precipitated a final time and dissolved in 300 11 (1 is used for micron) sterile water. Transformation of Agrobacterium strains by electroporation Broad host-range plasmids were introduced into A. radiobacter strains using electroporation. A. radiobacter strains were grown at 30°C in 1 1 flasks containing 200 ml DYT. Cells were harvested by centrifugation during exponential growth (A., nf) - 0.2 -0.5), washed three times with 25 ml ice-cold double-distilled water, resuspended in 2 ml 10% glycerol and stored on ice. Plasmid DNA (0.5 - 2.0 mg) was mixed with 0.2 ml washed cells which were immediately trans- ferred to an electroporation chamber and subjected to a high voltage pulse using a Bio-Rad Gene Pulser with the voltage set at 2.5 kV and the shunt resistance at 400-600ohms. Following electroporation, the cells were transferred to 1.0 ml DYT medium, shaken at 30 C for 1.5 h and plated out onto nutrient agar contain- ing gentamicin or kanamycin (30 and 100 lg 1 respectively) to select for transformants. Oligonucleotide synthesis. Oligonucleotides were synthesised using an Applied Biosystems 380B instrument. Amplification of DNA sequences using the polymerase chain reaction (PCR) Reactions were carried out using a standard mix of 100 ml volume which contained 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 πM MgCl« , two oligonucleotide primers (each at a concentration of 1.0 1M) , 2001M dNTP's, 2.5 units A plitaq DNA polymerase (Perkin Elmer Cetus) and lOOng template DNA. The temperature cycling conditions were varied to optimise product formation with each set of primers. However, in all cases some product was obtained using 25 cycles of the following sequence which is recommended by the supplier of the enzyme as a useful starting point on which to base a successful amplification protocol; 1 min at 94 C to melt strands, 1 min at 37 C to anneal primers and 2 min at 72 C for polymerisation. DNA sequencing DNA fragments were cloned into pUC18 for sequencing using the chain termination method. DNA sequencing kits containing T7 polymerase were purchased from U.S. Biochemical Corp and reactions were carried out using the manufacturer's protocols. Experiments Isolation of strains of A. radiobacter NCIB 11883 that hyperproduce periplasmic, sugar-binding proteins during carbon-limited growth. It has previously been reported that prolonged growth of A ; _ radiobacter NCIB 11883 in continuous culture under glucose limita¬ tion (D- 0.045h * ) resulted in the emergence of new strains which hyperproduced one of two periplasmic, glucose-binding proteins. A. radiobacter strain AR9 hyperproduced GBP2 and strain AR.18, which has been deposited as A. radiobacter NCIB 40018, hyperproduced GBP1. Further characterisation of the glucose-binding proteins revealed that GBP1 also binds D-galactose much more tightly than D-xylose wh: t the converse is true for GBP2 (Table 2) . When A_ diobacter NCIB 11883 was grown for a prolonged period under galactose or xylose limitation, strains were isolated that hyperproduced GBP1 or GBP2 respectively (Table 3) . Additional work concerning the mode of transport of the disaccharide lactose revealed that A. radiobacter NCIB 11883 also produces a periplasmic, lactose-binding protein (LBP) which can be released from cells using osmotic shock. Binding studies carried out with pure LBP indicated that it binds lactose with high affini¬ ty (Table 2). Furthermore, prolonged growth of A. radiobacter NCIB 11883 under lactose limitation resulted in the emergence of strains which hyperproduced LBP, and one of these, strain AR50, was select¬ ed for further study (Table 3). Hyperproduction of discrete proteins in response to sugar-lim¬ ited growth was also observed with a strain of A. tumefaciens which has been deposited as NCIB 40325. During growth on glucose this organism produced two proteins (termed "GBP1" and "GBP2") which were indistinguishable from GBP1 and GBP2 produced by ^ radiobacter as regards their size and immunological cross-reactiv¬ ity and appear to be identical proteins. Prolonged growth under glucose and galactose limitation resulted in the emergence of A. tumefaciens strains which hyperproduced "GBP2" and "GBP1" respec¬ tively (Fig. 1). it therefore seems to be a general rule that Agrobacterium strains which hyperproduce periplasmic, sugar-binding proteins can be obtained by growing the organisms in continuous culture under carbon limitation provided that the appropriate sugar (i.e. one that binds to the periplasmic protein with high affinity) is used as the growth-limiting substrate. Expression of sugar-binding proteins by hyperproducing strains of A. radiobacter during growth in carbon-limited continuous culture using different sugars as the growth-limiting nutrient. Under conditions of glucose-limited growth, GBP1 comprised approximately 30% of the total cell protein produced by A. radiobacter NCIB 40018. Given that glucose and galactose are good substrates for the GBP1 transport system (and bind to GBP1 in vitro; Table 3) it appeared that GBP1 was being induced by these sugars during carbon limitation to enable the organism to take up the substrates when their standing concentrations in the growth medium were extremely low. However, it turned out that A. radiobacter NCIB 40018 always produced GBPl during growth in carbon-excess batch culture on a wide range of carbon sources (although at a much lower level than during carbon-limited growth), indicating that the gene encoding GBPl was being expressed at a higher level than in the wild-type strain. Nevertheless, SDS-PAGE analysis of whole cells and measure¬ ment of glucose transport rates indicated that the levels of expression of GBPl varied according to the growth substrate used, indicating that synthesis of GBPl was being modulated via some form of catabolite repression. Taken together, these observations suggested that A. radiobacter NCIB 40018 would produce GBPl at high concentrations during growth on any carbon source provided that carbon-limited conditions (i.e. in continuous or fed-batch culture) were used to minimise the extent of catabolite repression that could be exerted by a given substrate. Using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) it was confirmed that GBPl was the major cellular protein produced by A. radiobacter NCIB 40018 during growth under carbon-limited, fed batch conditions under sucrose, glycerol and glucose limitation. Further experimentation revealed that A. radiobacter AR9 hyperproduced GBP2 during growth under galactose limitation even though this sugar binds weakly to GBP2 in vitro (Table 2) and is not taken up by the GBP2-dependent transport system. Similarly, strain AR50 produced large amounts of LBP during growth under galactose limitation even though galactose proved to be a poor substrate for the LBP-dependent lactose transport system. Growth of Agrobacterium strains under prolonged sugar limita¬ tion leads to the emergence of strains that hyperproduce the periplasmic binding proteins in response to carbon-limited growth per se. The DNA preceding the binding protein genes in the novel strains seems to contain a promoter which can be activated by , ' ar<*. carbon limitation, in which case the binding protein promoters could be fused to foreign genes to drive heterologous expression of proteins in Agrobacterium in response to a carbon-limited growth regime. Cloning of the GBPl promoter from A. radiobacter NCIB 40018. A 950bp region of the A. radiobacter NCIB 40018 chromosome containing the GBPl promoter and part of the GBPl structural gene was amplified using the polymerase chain reaction (PCR) with the following pair of oligonucleotide mixtures as primers: 5' primer (24mer, 16 redundancies): 5'-GGC TGC AGG [T,C]TG [A,C,G,T]GT CAT [A,C]TT CAT-3'. 3' primer (20mer, 64 redundancies): 5'-GC [C,T]TG [A,G]TC [A,G]TA [A,G]AA [A,G]AA [A,G]AA-3' This piece of amplified DNA was digested with Pstl to give a DNA fragment of approximately 850bp which was cloned into pUC18 to give pACl (Fig.2). The relevant parts of the DNA sequence are given in Table 1. Fusion of the GBPl promoter to the type III chloramphenicol acetyl transferase gen gene from Shigella. The GBPl promoter region and the start of the GBPl structural gene was amplified from pACl using PCR with the following pair of oligonucleotide primers: 5'-primer (24mer, 16 redundancies): 5'-GGC TGC AGG [C,T]TG [A,C,G,T]GT CAT [A,G]TT CAT-3' 3'-primer (25mer) 5 > .GCGAAATAATGGAATTCATATTCTC-3' A type III chloramphenicol acetyl transferase (CAT) was amplified from pUC18:IM3:Clal (Murray et al. , 1988) using PCR with the following pair of oligonucleotide primers. 5'-primer (28mer) : 5'-GGGATCGATTATGAATTCTACAAAATTT-3' 3'-primer (32mer) : 5' -CGGCTTAATAATATCCTAGGAAGGCGTTTTCC-3' PCR products containing the GBPl promoter and the promoterless CAT gene were digested with Pstl/Ecol and EcoRI/BamHI respectively and ligated into pUC18 which had been cut with Pstl/BamHI. E. coli JM105 was transformed with the ligation products and plated onto nutrient agar which contained atp cillin and chloramphenicol (both at a concentration of 501g ml ) to select for the vector and insert respectively. Restriction-enzyme mapping of plasmids isolat¬ ed from ten chloramphenicol-resistant transfor ants confirmed that GBPl promoter and CAT gene had been fused successfully as did DNA sequencing of the GBPl promoter/CAT junction in the construct. The fusion was constructed such that the first three amino acid resi- dues of CAT were replaced by the first three residues of the precursor form of GBPl. Furthermore, the CAT gene was cloned in the opposite orientation to the lacZ promoter on the vector. The vector containing the GBPl promoter/CAT gene cartridge was termed pCRl (Fig. 3). Construction of broad host-range vectors containing the GBP1/CAT cartridge. pCR3 contains a single origin of replication (ColEl) and therefore could not be used to examine expression of the CAT gene in strains of A. radiobacter. The whole cartridge was therefore transferred into two broad host range vectors, pBIN19 (Bevan, 1984) and pGEl, both of which can replicate in Agrobacterium and are maintained at a low copy number. pGEl also contains a ColEl origin of replication which enables it to be maintained in E. coli at high copy number. ' i ' : The GBP1/CAT cartridge was excised from pCRl using HindiII and BamHI, purified using preparative electrophoresis, and ligated into the broad host range vector pBIN19 which had been cut with Hindlll and BamHI. Ligation products were used to transform E. coli SK1592 and clones exhibiting resistance to kanamycin (encoded by pBIN19) and chloramphenicol were obtained following growth on nutrient agar containing both these antibiotics at a concentration of 501gml " . Restriction mapping indicated that the CAT cartridge had been inserted into pBIN19 yielding pCR2, which contained the CAT gene cloned in the same orientation as the lacZ promoter (Fig. 4) . In order to insert the CAT cartridge into pGEl it was neces¬ sary to digest pCRl completely with Hindlll and partially with EcoRI so as to release a 1.4kb fragment containing Hindlll/EcoRI sticky ends. The DNA fragment containing the intact CAT cartridge (i.e. the GBPl promoter fused to the CAT structural gene) was separated from other digestion products using preparative electrophoresis and ligated into pGEl which had been cut with Hindlll and EcoRI. Ligation products were used to transform E. coli SK1592 and clones containing the CAT cartridge were selected by plating transformants onto nutrient agar containing gentamicin and chloramphenicol (10 and 501g ml respectively) . Clones that were resistant to both gentamicin and chloramphenicol contained plasmid pCR3 (Fig. 5). Expression of active CAT protein in E. coli containing pCRl using the GBPl promoter. E. coli JM105 containing pCRl was grown at 37°C in 200ml M9 medium using glucose as the carbon source. After 16h incubation the culture was used to inoculate a 3 1 Biotec fermenter containing 3 1 of Medium 1. Growth occurred exponentially until the initial glucose present was exhausted, at which time additional glucose was fed into the reactor a constant rate of 2.6 gh for 43 h so as to maintain glucose-limited, fed-batch growth: The biomass increased linearly after the initial period of exponential growth and the expression of CAT increased significantly during the extended period of glucose-limited growth (Fig. 6). The increase in the amount of CAT could also be observed from SDS-PAGE analysis of whole cells (Fig. 7) . Expression of active CAT protein from the GBPl promoter in A. radiobacter strain NCIB 11883 containing pCR2. Plasmid pCR2 was introduced into A. radiobacter NCIB 11883 using electroporation. The plasmid-containing strain was grown in a shake-flask containing 200 ml Medium 3 for 16 h after which time the culture was used to inoculate a Biotec fermenter containing 3 1 Medium 2. The culture grew exponentially for 14 h at which point glucose was fed into the fermenter at a constant rate of 6 gh for the next 43 h. The bacterial biomass increased linearly to a final value of 13.2 g dry wt 1 and the use of a continuous feed ensured that growth was glucose-limited during this time (i.e. there was negligible free glucose in the culture) . As expected, the level of expression of CAT was influenced by the amount of available glucose present in the culture. During exponential growth in the presence of excess glucose CAT activity was found to be 1.4 lmole [mg protein] min but this increased during the period of glucose- limited growth to reach value of 3.6 lmoles [mg protein] min (Fig. 8). The increase in CAT activity correlated with the increase in CAT protein (Fig. 9). This demonstrates that the GBPl promoter from A radiobacter NCIB 40018 can be used to the control expression of heterologous proteins as claimed above. Considerably higher levels of expression are expected if the period of carbon-limited growth was extended by increasing the glucose feed rate periodically or by feeding glucose at an exponentially increasing rate. Controlled expression of CAT from the GBPl promoter in A. radiobacter NCIB 11883 containing pCR.3. Plasmid pCR3 was introduced into A. radiobacter NCIB 11883 using electroporation. The plasmid-containing strain was grown in a shake-flask containing 200 ml Medium 3 for 16 h after which time the culture was used to inoculate a Biotech fermenter containing 3 1 Medium 2. Growth occurred exponentially for 14 h after which time glucose was fed into the culture at a constant rate of 6gh for the next 43 h.The bacterial biomass then increased to a final value of 13.7 gl (Fig. 10) over the period of glucose-limited growth. -1 -1 CAT activity of approximately 2.8 Imole [mg protein] min was measured during exponential growth when there was excess glucose in the culture. CAT activity increased significantly during the period of glucose-limited growth, reaching a final value of 8.3 Imoles [mg protein] min . The increased levels of CAT protein correlated with the measured enzyme activity (Fig. 11) . These findings confirmed that the GBPl promoter from A^ radiobacter NCIB 40018 can be used to control expression of heterologous proteins by imposing carbon-limited growth using a fed-batch system. Considerably higher levels of expression could be obtained using an extended period of carbon- limited growth. This could be accomplished either by increasing the glucose feed rate at intervals or by using an exponentially-increasing feed rate to maintain a low but constant growth rate. Use of the GBPl signal sequence to secrete heterologous proteins into the periplasm of A. radiobacter NCIB 11883. Using PCR methodology the GBPl promoter and signal sequence was fused to part of the b-lactamase gene from pUC18 such that secretion into the periplasm and correct processing of the GBPl signal sequence would yield the mature b-lactamase. The GBPl promoter/ GBPl signal sequence/ modified b-lactamase gene fusion was cloned into the broad host-range vector pLAFR3 (Staskawicz et al. , 1987) and introduced into A. radiobacter NCIB 11883. Adding the GBPl signal sequence to the mature b-lactamase resulted in secretion and processing of the protein in A. radiobacter NCIB 11883 as cells containing the pLAFR3-based secretion cartridge were resistant to 500 mg ml ampicillin whereas the strain is usually killed at 50 mg ml " . It is known that periplasmic proteins (including GBPl) can be released by osmotic shock, hence the production and recovery of heterologous proteins in A. radiobacter can be improved by fusing the foreign gene to the Agrobacterium promoter sequence plus the GBPl promoter and signal sequence. Expression of the LBP gene from AR60 in A. tumefaciens and E. coli and secretion of the LBP into the periplasm. Genomic DNA A. radiobacter strain AR60 was digested with a variety of restriction enzymes and subjected to Southern blotting 32 analysis using a P- labelled mixed oligonucleotide probe [5'-ATT(CA) TGG TGT(C) TGG GAT(C) CC- 3'] which was modelled on the sequence -IWCWDP- found near the N-terminus of the mature protein. The probe hybridised to an 11.5kb fragment when genomic DNA was digested to completion with Hindlll. The 11.5kb DNA was purified from a low-melting point agarose gel, lig ad into pBIN19 which had been cut with Hindlll in the multiple cloning site, and transformed into E. coli HB101. Clones expressing LBP and its accessory trans- port proteins were obtained by plating transformants onto minimal medium containing lactose as sole carbon source [E. coli HB101 cannot grow on lactose as it lacks the lactose permease (lacY gene product)] and kanamycin (50mg ml-1) to select for the vector. Several clones capable of growing on lactose were obtained and these all expressed LBP as determined from SDS-PAGE analysis of cells and Western blotting analysis using antiseru raised against pure LBP (Fig. 12). Furthermore, LBP could be released from E. coli HB101 using osmotic shock indicating that it was being secreted into the periplasm. The pBIN19 vector containing the 11.5kb insert was digested with Pstl and a 3.75kb DNA fragment containing the oligonucleotide binding site plus 1.4 kb of the pBIN19 vector was subcloned into the vector pUC19 for sequencing. The signal sequence of LBP was deduced from the DNA sequence of the subcloned fragment of the LBP gene that encoded the amino terminus of the protein (Table 4) . The DNA sequence data also revealed that the LBP gene had been cloned in the opposite orienta¬ tion to the lacZ promoter in pBIN19. These results indicate that LBP from AE.50 is expressed effi¬ ciently from its own promoter in E. coli and secreted into the periplasm. The LBP promoter could be therefore be used to drive expression of heterologous proteins in E. coli and the LBP signal sequence could be used to direct foreign proteins into the periplasm. LBP was also expressed and processed correctly in ^ tumefaciens which did not contain an LBP-based lactose transport system, hence an expression and/or secretion system based on the LBP promoter/signal sequence will function in A. tumefaciens. References Bevan, M. (1984). Nucleic Acids Research 12, 8711-8721. Birnboim, H. C. & Doly, J. (1979). Nucleic Acids Research 7, 1513. Cornish, A. Greenwood, J.A. & Jones, C.W. (1988). Journal of General Microbiology 134, 3099-3110. Chow, L.T., Kalman, R. & Kamp, D. (1977). Journal of Molecular Biology 113, 591-609. Linton, J.D., Evans, M.W. , Jones, D.S. & Gouldeney, D.G. (1987). Journal of General Microbiology 133, 2961-2969. Maniatis, T. , Fritsch, E.F. & Sambrook, J. (1982). Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. Murray, I.A., Hawkins, A.R. , Keyte, J.W & Shaw, W.V. (1988). Biochemical Journal 252, 173-179. Shaw, W.V. (1975). methods in Enzymology XLIII, 737-755. Staskawicz, B. , Dahlbeck, D. Keen, N. & Napoli, C. (1987). Journal of Bacteriology 169, 5789-5794. Towbin, H. , Staehlin, T. & Gordon, J. (1979). Proceedings of the National Academy of Science 76, 4350-4354. Yanisch-Perron, C. , Vieira, J & Messing, J. (1985). Gene 33, 103-119. Table 1. SEQUENCE TYPE: Nucleotide with corresponding protein SEQUENCE LENGTH: 453 base pairs STRANDEDNESS:single TOPOLOGY:linear MOLECULE TYPE:genomic DNA ORIGINAL SOURCE ORGANISM:Agrobacterium radiobacter NCIB 40018 FEATURES:from 344-347bp RBS (ribosome binding site) from 355-426bρ signal peptide from 427-453 N-terminus of mature peptide from 10-354bp promoter PROPERTIES:promoter region and part of coding region of GBPl, a periplasmic glucose-binding protein. GTGGCTCATC TTCAATCCGG CCCGCAGCAA AGGGTTGTCT TCAAAGACGA AAAGGGCATC 60 CGTTGTTTGC GCCTGCTCAT ACACTTTTTG GTTGTTCATT CGTGGTTTTA TACCCCTCAT 120 CGGTTTTTGT ATCGATTTAA TATATCATAA TTGATATATG GAAACTGATT TATTCTATTT 180 GACAGTTATA TGGATTCAGA ATAATTTGCG GAATGCCGGG GAGGAGCGGC ACGTGTTCCA 240 TTTCGCAATT GCGAAATCGG GGTGAGCGCA ATGCGGGCTT CTTCCTCTGA CGGCATGAAT 300 TTCATGGTGC ACCGCAGCAT GCGGCTCCAG AGAAGGGCTC AAGGGAGAGA GTTA ATG 357 Met AAG TCC ATT ATT TCG CTG ATG GCA GCT TGT GCC ATC GGT GCT GCT TCT 405 Lys Ser lie lie Ser Leu Met Ala Ala Cys Ala lie Gly Ala Ala Ser -20 -15 -10 TTC GCA GTG CCG GCT " TTC GCA CAG GAC AAG GGT TCC GTT GGT ATC GCC 453 Phe Ala Val Pro Ala Phe Ala Gin Asp Lys Gly Ser Val Gly He Ala -5 1 Table 2 Pro erties of eri lasmic, su ar-bindin roteins isolated from Tab1e 3. Strains of Agrobacterium that hyperproduce periplasmic, sugar-binding protein ND - extent of hyperproduction not quantified but evident from SDS- PAGE analysis of whole cells. Increase in Limitation Major binding protein Parent Strain for strain sugar-binding content over organism isolated selection protein wild-type (% total protein) A.radiobacter AR9 glucose GBP2 15 -> 40% " K. - 11883 AR9a xylose GBP2 ND A.radiobacter glucose GBPl 4 -> 35 NCIB40018 ARlδa galactose GBPl ND AR50 lactose LBP 8 ->30 A.tumefaciens AT9 glucose "GBP2" ND AT18 galactose "GBPl" ND Table 4. SEQUENCE TYPE: Nucleotide with corresponding protein SEQUENCE LENGTH: 234 base pairs STRANDEDNESS: Single TOPOLOGY: Linear MOLECULAR TYPE: Genomic DNA ORIGINAL SOURCE ORGANISM: Agrobacterium radiobacter NCIB 11883 strain AR50 FEATURES: from 1 to 132bp promoter from 123 to 127 ribosome binding site RBS from 133 to 216 signal peptide from 217 onwards N-terminus of mature protein PROPERTIES: Periplasmic, lactose-binding protein (LBP) TCCGACACTA CGCAATGCAC AATATTTTAG TAAAATTTTC TTGACTGTCC TCCGATTTTG 60 GAAATAGTGG CCATGAGGCA GGGAAAGAGC GTGCAGCGGT TAACGTCATA ATGGAGGAAC 120 GC ATG GAT TAT TCT CGC TTG CTC AAG CGC TCC GTT TCG 158 Met Asp Tyr Ser Arg Leu Leu Lys Arg Ser Val Ser -25 -20 GCT GCG CTG ACG GCT GCC GCA CTG TTG TGC AGC ACG GCG GCC TTT GCC 206 Ala Ala Leu Thr Ala Ala Ala Leu Leu Cys Ser Thr Ala Ala Phe Ala -15 -10 -5 GGC GAA GTC ACC ATC TGG Gly Glu Val Thr He Trp 1 5

Claims: Claims : 1. A DNA promoter sequence which is present in the DNA sequence as given in Table 1. 2. A DNA promoter sequence according to claim 1, which is present in the DNA sequence as given in Table 1 between base number 9 and base number 355, preferably between base nvimber 140 and base number 355. 3. A DNA promoter sequence according to claim 2, which is present in the DNA sequence as given in Table 1 between base number 200 and base number 355 preferably between base number 260 and base number 355. 4. A DNA promoter sequence which is present in the DNA sequence as given in Table 2. 5. An expression vector comprisir . a promoter-gene combination, preferably a heterologous promoter-gene combination, in which the promoter is activated by carbon limitation, preferably carbohydrate limitation. 6. An expression vector according to claim 5, which additionally comprises a secretion signal, preferably a secretion signal which is homologous with the promoter. 7. An expression vector according to claim 6, in which the promoter is derived from DNA which controls the expression of a periplasmic binding protein. 8. An expression vector according to claim 7, in which the promoter after introduction into a host is activated during growth of the host on carbon sources other than those bound by the binding protein. 9. An expression vector according to claim 7, wherein the promot¬ er is derived from a mutant-strain of a microorganism, the mutation having occurred after cultivation of the microorganism under nutri¬ ent-limited conditio* preferably carbon-limited conditions. 10 An expression vector according to claim 8, wherein the micro¬ organism is a strain of Agrobacterium, preferably Agrobacterium radiobacter or Agrobacterium tumefaciens, more preferably Agrobacterium radiobacter, still more preferably Agrobacterium radiobacter deposited as NCIB 40018 or NCIB 40325. 11. An expression vector according to claim 5, wherein the promot¬ er is is a promoter according to any one of claims 1 to 4. 12. A host containing a vector according to any one of claims 5 to 11, wherein the host is a micro-organism, preferably a bacteria, more preferably an Agrobacterium strain or an Escherichia strain, still more preferably Agrobacterium radiobacter, Agrobacterium tumefaciens or Escherichia coli. 13. A method for the production of polypeptides, which comprises cultivating a host according to claim 12, preferably under carbon- limited conditions. 14. A method according to claim 13, which additionally comprises the step of releasing the secreted polypeptides, preferably of releasing the poiypeptide to the periplasm in case the host is a Gram-negative micro-organism, optionally followed by an osmotic shock. 15. Artificially constructed DNA comprising the DNA promoter se¬ quence according to any one of claims 1 to 4. SUBSTITUTE SHEET

Inventors: BETTERIDGE PETER RICHARD, CORNISH ALEXANDER, GREENWOOD JACQUELINE ANNE, JONES COLIN WILLIAM, LINTON JOHN DUDLEY, WILLIAMS STEVEN GERAINT, BETTERIDGE, PETER, RICHARD, CORNISH, ALEXANDER, GREENWOOD, JACQUELINE, ANNE, JONES, COLIN, WILLIAM, LINTON, JOHN, DUDLEY, WILLIAMS, STEVEN, GERAINT
Applicants: SHELL INT RESEARCH, SHELL INTERNATIONALE RESEARCH MAATSCHAPPIJ B.V.
Application year: 1990
Main classification: C12N15
IPC codes: C07K14, C12N1, C12N9, C12N15, C12P21, C12R1