A leader sequence to promote the secretion of gene products

The present invention relates to fragments of specific . deoxyribonucleotide sequences that promote the secretion of gene products from cells and in particular to recombinant DNA transfer vectors that contain these fragments.

Recent developments in biochemistry have led to the construction of recombinant DNA transfer vectors in which, transfer vectors, for example plasmids, are made to contain exogeneous DNA. In some cases the recombinant incorporates heterologous DNA that codes for polypeptides that are ordinarily not produced by the organism susceptible to transformation by the recombinant vehicle.

In its basic outline a method of endowing a micro organism with the ability to synthesise a new protein involves three. general steps:

• (a) isolation and purification of the specific gene or nucleotide sequences containing the genetically coded information for the amino acid sequence of the desired protein or polypeptide,
• (b) recombination of the isolated gene or nucleotide sequence with an appropriate transfer vector, typically DNA of a bacteriophage or plasmid to form a recombinant transfer vector that codes, in part, for the production of the desired protein or polypeptide,
• (c) transfer of the vector to the appropriate micro organism and selection of a strain of the recipient micro o rganism containing the desired genetic information.

Provided the gene or nucleotide sequence expresses its protein or polypeptide in the chosen micro organism, growth of the micro organism should then produce the desired protein or polypeptide in significant quantities.

Once the micro organism has been cultured, the protein or polypeptide must be isolated from the undesired materials. This step is considerably facilitated if the majority of the desired protein or polypeptide is present in the culture medium and/or the periplasmic space of the micro organism. In other words purification may be performed in a more efficient manner if, once expressed, the protein or polypeptide passes through the cell membrane and out of the cytoplasm.

The passage of the protein or polypeptide through the cell membrane is desirable for two main reasons. First the desired protein or polypeptide will generally be foreign to the micro organism in which it is expressed. In many cases, therefore, it will be quickly broken down by proteolytic enzymes etc in the cells cytoplasm and will, subsequently, have a short half life within the cell. By transferring the protein or polypeptide out of the cytoplasm soon after expression the stability of the protein or polypeptide will be greatly increased. Second the number of unwanted genetic materials and products (from which the desired protein or polypeptide must be isolated) will be far greater in the cell's cytoplasm than in the culture medium and/or in the cell's periplasmic space. It can be seen that on both of the above counts the transfer of the protein or polypeptide through the cell membrane and out of the cytoplasm will greatly facilitate protein or polypeptide isolation.

One way in which the secretion of gene products from the cell's cytoplasm may be promoted is to produce, within the cytoplasm, a preprotein or prepolypeptide in which the desired protein or polypeptide is preceded by a signal polypeptide. The predominantly hydrophobic signal polypeptide directs the desired protein or polypeptide to the cell's periplasmic space, where the signal peptide is removed as the desired protein or polypeptide traverses the cell membrane.

Many of the known signal peptides contain cysteine residues. These residues have been found to react in the cell membrane and thereby inhibit the efficient transfer of the desired gene product out of the cell.

It is the primary object of the present invention to provide recombinant DNA transfer vectors containing a leader sequence polynucleotide that codes for a signal peptide that is cysteine free. Other objects and advantages of the present invention will become apparent from the following description thereof.

According to the present invention there is provided a recombinant DNA transfer vector comprising a leader sequence polynucleotide coding for signal polypeptide of formula I,

The transfer vector may be a bacteriophage or, which is preferred, a plasmid.

Preferably the majority of the codons in the nucleotide sequence are those preferred for the expression of microbial genomes. Suitable codons are listed in UK 1,568,047 and UK 2007675A, and these publications are therefore incorporated herein by reference.

In one preferred embodiment of the present transfer vector the nucleotide sequence has formula II

The nucleotide sequence coding for the signal polypeptide (the leader sequence poly nucleotide) will preferably be downstream of and in reading phase with a bacterial or a yeast promoter and a prokaryotic ribosome binding site in the transfer vector. Moreover the leader sequence polynucleotide will either be upstream of an insertion site for a structural gene or, which is preferred, will be upstream of and in reading phase with a structural gene coding for a desired protein or polypeptide. Preferably the gene codes for a eukaryotic, particularly a mammalian, protein pr polypeptide.

The structural gene may code, for example, for such eukaryotic proteins as human growth hormone, human insulin or human chorionic somatomammotropin. Alternatively it may code for such prokaryotic proteins as E.coli β-galactosidase or Pseudomonas carboxy peptidase G₂ (CPG₂) (Carboxypeptidase G₂ is an enzyme, produced by Pseudomonas species strain RS-16, that has application in cancer chemotherapy.

It is a Zn ²⁺ containing dimer of 2 x 42,000 daltons and has high affinities (Em values of 10^-5 or 10^-6M) for both 5-methyltetra- hydrofolate, the predominant ciculatory form of folate in mammals and for the folic acid antagonist methotrexate (MTX), which is widely used in cancer chemotherapy. The enzyme may be used directly for the plasma depletion of reduced folates, essential as co-factors in purine and particularly in pyrimidine biosynthesis. CPG₂ has been shown to inhibit the development of the Walker 256 carcinoma in vivo and to remove MIX from circulation in patients where prolonged exposure to high doses of MTX leads to toxicity).

Examples of transfer vectors according to the present invention that code for _CPG2 are pNM1, pNM111, p_NM14, pNM21, pNM22, pNM31, pNM32 and pLEC3.

The promoter is preferably a high expression bacterial or yeast promoter for the structural gene in a variety of hosts. The particular choice of promoter will depend on the microorganism to be transformed. For example the transformation of E.coli will generally be effected by a transfer vector in which an E.coli promoter controls the expression of the structural gene. Examples of E.coli promoters are those present in the plasmids pBR 322 and pA^T 153. By contrast, the transformation of Pseudomonas species will generally be effected by a transfer vector in which a Pseudomonas promoter controls the expression of the structural gene. Examples of Pseudomonas promoters are those present in the plasmid pKT 230 or Pseudomonas chromosomal DNA.

In order to express the structural gene the present transfer vector will be transformed into a suitable microorganism. According to a further aspect of the present invention therefore there is provided a microorganism transformed by a recombinant DNA transfer vector according to this invention. The microorganism will preferably be a bacterium or yeast in which high expression of the structural gene, within the transfer vector, occurs. Depending on the choice of promoter the microorganism may be a strain chosen from one of the following bacteria E.coli, Pseudomonas and Bacillus or the yeast Saccharomyces cerevisiae.

Having transformed the microorganism, the protein or polypeptide, for which the structural gene codes, may then be expressed by culturing the transformed microorganism in a culture medium. It is the primary advantage of the present invention that culturing the transformed microorganism affords a preprotein or prepolypeptide in which the desired protein or polypeptide is preceded by the present signal polypeptide. This means that soon after expression the signal polypeptide directs the desired protein or polypeptide to the cell's periplasmic space, where the signal polypeptide is removed as the desired protein or polypeptide traverses the cell membrance. Since the present signal polypeptide is free of cysteine residues the desired gene product will be efficiently secreted through the membrane.

The present transfer vectors may be prepared by any of the methods that are well known in the recombinant DNA art. For example the leader sequence poly nucleotide may be synthesised by the modified triester method of X.Itakura etal, JACS, 1975, 97, 7327 or by the improved oligodeoxynucleotide preparation described in UK 2007675A. The disclosure of both of these references is incorporated herein by reference. The synthesised polynucleotide may then be inserted in a transfer vector, preferably a plasmid. In the transfer vector it will preferably be downstream of and in reading phase with a bacterial or a yeast promoter and a prokaryotic ribosome binding site. The leader sequence polynucleotide should also be either upstream of a structural gene insertion site or upstream of and in reading phase with a structural gene.

Alternatively, DNA fragments containing the leader sequence polynucleotide may be obtained from natural sources, in particular from the chromosomal DNA of Pseudomonas species strain RS-16. In this particular case a polynucleotide (formula II above) coding for the present signal polypeptide immediately precedes a structural gene coding for CPG₂. A number of the DNA fragments containing this leader sequence polynucleotide may therefore be recognised by their ability, on insertion into a plasmid and transformation of a microorganism by the resultant recombinant vector, to enable a microorganism to grow on folate. Examples of such recombinant transfer vectors that contain both a polynucleotide coding for the present signal polypeptide(formula II above) and a structual gene coding for CPG₂ are pNM1, pNM111, pNM14, pNM21, pNM22, pNM31, phM32 and pLEC3. Of course, once a Fol⁺ recombinant vector has been obtained in this way it may be subcloned to afford alternative vectors (either Fol or Fol-) that also contain a polynucleotide coding for the present signal polypeptide.

Once a suitable DNA fragment has been isolated it may then be inserted in a transfer vector, preferably a plasmid. In the transfer vector the leader sequence polynucleotide on the inserted fragment should be downstream of and in reading phase with a bacterial or a yeast promoter and a prokaryotic ribosome binding site. The leader sequence polynucleotide should also be either upstream of a structural gene insertion site or upstream of and in reading phase with a structural gene.

The structural gene for insertion downstream of and in reading phase with the present leader sequence polynucleotide may be obtained, for example, by the synthetic methods mentioned above (this is particularly useful for the preparation of genes coding for small proteins, such as human growth hormone, insulin and human chorionic somatomammotropin.) Alternatively the structural gene may be prepared from m=RNA by the use of the enzyme reverse transcriptase or may be isolated from natural sources (chromosomal DNA).

An example of the latter method is the isolation of DNA fragments containing a polynucleotide sequence (shown in Table 1) coding for the enzyme CPG₂ (amino acid sequence also shown in Table 1) from Pseudomonas species strain RS-16 chromosomal DNA. Examples of plasmids containing a CPG₂ structural gene, as well as a polynucleotide coding for the present signal polypeptide (formula II above), are pNM1, pNM111, pNM14, pNM21, pNM22, p_NM31, pNM32 and pLEC3.

Once prepared or isolated the Leader sequence polynucleotide and the structural gene will be inserted into a transfer vector, preferably a plasmid, to form a recombinant DNA transfer vector according to the present invention. The insertion step or steps will preferably be effected by one of the well known techniques in this art that employ restriction endonucleases, see for example the methods discussed in UK 2090600A, the disclosure of which is incorporated herein by reference. The choice of transfer vector will be determined by the microorganism in which the leader sequence polynucleotide and structural gene are to be expressed. Generally the transfer vector will be a cloning vehicle that is suitable for transforming the chosen micro-organisms and that displays a phenotypical characteristic, such as antibiotic resistance, by which the recombinant transfer vectors may be selected. Thus, if the micro-organism is to be E-coli, then suitable transfer vectors will be the E-coli plasmids pBR322 and pAT153. Alternatively, if the micro-organism is to be Pseudomonas,

Amino acids 1 to 22 are the present signal polypeptide Amino acids 23 to 415 are the CPG₂ structural gene

NB The leader sequence polynucleotide is the preferred polynucleotide of formula II. then a suitable transfer vector will be Pseudomonas pkT230.

The present recombinant DNA transfer vectors, micro-organisms transformed by the present recombinant DNA transfer vectors and processes for the preparation of said vectors and micro-organisms will now be described by way of example only, with particular reference to the Figures in which:

• Figure 1 is a restriction enzyme cleavage site map of pNM1,
• Figure 2 is a restriction enzyme cleavage site map of pNM111,
• Figure 3 is a restriction enzyme cleavage site map of pNM14,
• Figure 4 is a restriction enzyme cleavage site map of pNM21,
• Figure 5 is a restriction enzyme cleavage site map of pNM22, and
• Figure 6 illustrates the process for the preparation of a recombinant plasmid containing both the present leader sequence polynucleotide and the β-Galactosidase structural gene, and
• Figure 7 is a restriction enzyme cleavage site map of pLEC3.

Materials and Methods

Bacterial strains and plasmids

The bacterial strains used were Escherichia coli W5445 (pro leu thi thr^o sup E44 lac Y ton A r^- m^- Str^r) Pseudomonas putida 2440 (r^-) and Pseudomonas sp strain RS-16. The plasmids employed were pBR322 (F Bolivar et al Gene, 1977, 2, 95), pAT153 (A J Twigg et al, Nature, 1980, 283, 216) and pKT230 (M Bagdasarin et al, Gene 1981, 16, 237) and pROG5 (R.F.Sherwood et al, The Molecular Biology of Yeast, 1979 Cold Spring Harbor Publications).

Media and culture conditions

E.coli was routinely cultured in L-broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl). Solidified medium (L-agar) consisted of L-broth with the addition of 2% (w/v) agar (Bacto-Difco). Antibiotic concentrations used for the selection of transformants were 50 µg/ml ampicillin, 15 µg/ml tetracycline and 30 µg/ml kanamycin. In the case of E.coli these were conducted in 2YT liquid medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) containing 1% glucose, and 0.05% folate where appropriate. The pseudomonads were grown in a minimal salts solution consisting of per litre: MgS0₄, 0.05g; CaCl₂, 2H₂0, 0.05g; FeSO₄.7H₂O, 0.005^g; MnSO₄, 0.0015^g; Na₂Mbo₄, 2H₂0, 0.0015^g; KH₂PO₄; 5^g; K₂HPO₄:3H₂O, 12g; glutamate, 10g. The minimal medium employed for E.coli was M9 medium (J Miller, Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972).

Purification of DNA

Plasmids were purified from chloramphenicol amplified cultures (D B Clewell, J Bacteriol, 1972, 110, 667) by Brij-lysis (D B Clewell et al, Proc Natl Acad Sci, USA, 1969, 62, 1159) and subsequent caesium chloride-ethidium bromide density gradient centrifugation (A Colman et al, Eur.J Biochem, 1978, 91, 303). A rapid, small scale plasmid isolation technique (Burnboim et al, Nuc.Acids Res, 1979, 7, 1513) was also employed for screening purposes. Chromosomal DNA from the donor Pseudomonad strain (RS-16) was prepared essentially as described by J Maxmar, J.Mol.Biol, 1961, 3, 208.

Restriction, ligation and transformation methods

Restriction endonucleases and DNA ligase were purchased from Bethesda Research Laboratories and used in the buffers and under the conditions recommended by the supplier. Transformation of E.coli was essentially as described by S N Cohen et al., Proc.Natl.Acad.Sci., USA, 1972, 69, 2110, while Ps.putida was transformed by the method of M Bagdasarian and K N Timmis, Current Topics in Microbiology and Immunology, Eds P H Hofschneider and W Goebel, Springer Verlag, Berlin, 1981, p 47.

Agarose gel electrophoresis

Digests were electrophoresed in 0.8% agarose slab gels (10 cm x 20 cm x 0.5 cm) on a standard vertical system (Raven), employing Tris-borate-EDTA buffer. Electrophoresis of undigested DNA was at 125V, 50 mA for 3 hours, while digested DNA was electrophoresed at 15V, 10 mA for 16 hours. Fragment sizes were estimated by comparison with fragments of λDNA digested with HindIII and λDNA cut with both HindIII and EcoRI. Fragments were isolated from gels using electroelution (M W McDonnell et al, Proc. Natl.Acad.Sci, USA, 1977, 74, 4835).

Determination of carboxypeptidase G₂ activity

Bacteria were grown in 1 litre batch culture and 100 ml samples taken at various stages in the growth phase. Samples were cooled on ice, centrifuged at 13,000 x g for 10 minutes and resuspended and frozen in 5 ml of 0.1 M Tris HCl, pH 7.3 containing 0.2 mM ZnSO₄. The cells were disrupted using a MSE Ultrasonic Disintegrator (150 W) at medium frequency, amplitude 2, for three 30-second intervals on ice. Cell debris was removed by centrifugation at 10,000 x g for 5 minutes.

CPG₂ activity was determined after J L McCullough et al, J.Biol.Chem, 1971, 246, 7207. A.lml reaction cuvette containing 0.9 ml of 0.1 M Tris-HCl, pH 7.3 plus 0.2 mM ZnSO₄ and 0.1 ml of 0.6 mM methotrexate was equilibrated at 37°C. Enzyme extract was added to the test cuvette and the decrease in absorbance at 320 nm measured using a Pye-Unicam SP1800 double-beam spectrophotometer. Enzyme activity per ml extract was calculated as Δ 320 nm absorbance/min divided by 8.3, which is equivalent to the hydrolysis of 1 µmol of MTX/min at 37°C. Protein concentration was determined by the method of M M Bradford, AnalBiochem, 1976, 72, 248.

Cell fractionation techniques

Bacterial cultures were grown in the low phosphate medium of H C Neu and L A Heppel, (J Biol Chem, 1964, 240, 3685), supplemented with 100 µg/ml ampicillin, to an OD₄₅₀ = 1.0. 40 ml of culture was centrifuged at 5000 g for 10 min, washed in 5 ml of 10 mM Tris-HC1 pH 7.0, and resuspended in 0.9 ml 0.58 M sucrose, 0.2 mM DTT, 30 mM Tris-HCl pH 8.0. Conversion to spheroplasts was achieved by the addition of 20 µl of lysozyme (2 mg/ml), 40 µl 0.1 M EDTA, and incubation at 23⁰ for 10 min (HC Neu et al, J Biol Chem, 1964, 239, 3893). The spheroplasts were placed on ice and 0.1 ml of 30% (w/v) BSA added, followed by 5 ml of sucrose-tris buffer. Sedimentation of the spheroplasts was achieved by centrifugation at 5000g for 10 min and the supernatant retained as the 'periplasmic' fraction. The pellet was resuspended in 5 ml 10 mM Tris-HCl, 0.2 mM DTT pH 7.0 and and sonicated at 20 Kc/sec, 2 Amps for 15 sec. Remaining whole cells were removed by centrifugation at 1000 x g for 10 min. Centrifugation at 100000 x g for 1 hr, at 4°C, separated the soluble (cytoplasmic) proteins from the particulate (membrane-bound) proteins. The membrane pellet was resuspended in 1 ml of 10mM Tris-HCl, 0.2 mM DTT, pH 7.0.

CPG₂ was assayed as described. Alkaline phosphatase was assayed according to J Miller, Experiments in Molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972, NADH oxidase according to M J Osborn et al, j Biol Chem, 1972, 247, 3962 and glyceraldehyde - 3 - phosphate dehydrogenase after K Suzuki et al, FEMS, 1971, 13, 217.

Example 1

Preparation of recombinant plasmid pNM1 (A plasmid containing both the present leader sequence polynucleotide and the CPG₂ structural gene)

To isolate the gene for carboxypeptidase G₂ together with the leader sequence polynucleotide chromosomal DNA prepared from the Pseudomonas host (strain RS-16) was partially digested with Sau3A and fragments of between 6-8 Md isolated from agarose gels by electroelution. The 'sized' DNA was ligated with alkaline phosphatase treated BamHl cut pBR322, transformed into E.coli W5445, and Ap^r transformants selected. Of the 3,500 Ap^r colonies obtained, approximately 70% were TC^s. Utilisation of a rapid plasmid isolation technique on 50 Ap^r TC^s transformants demonstrated that 90% of the gene bank harboured plasmids of the expected size. As a further check on the authenticity of the gene bank, the individual clones were screened for the acquisition of a Leu phenotype. Two such clones were identified. Both carried a plasmid capable of transforming leuB (B-isopropylmalate dehydrogenase) E.coli mutants to prototrophy.

Acquisition of a functional CPG₂ gene should enable E.coli to utilise folic acid as a carbon source. The 2,400 gene bank clones were screened for the ability to grow on minimal medium containing folate as the sole source of carbon (ie Fol⁺). A single Fol⁺ clone was detected and shown to harbour a plasmid capable of transforming plasmid-minus W5445 to the Fol⁺ phenotype. Classical restriction mapping of this plasmid (pNMl) was undertaken which revealed the presence of a 5.9 Md insert of pseudomonad DNA within pBR322. The restriction enzyme clearage site map of pNMl is given in Figure 1. The nucleotide sequence of the leader sequence polynucleotide and the CPG₂ structural gene is given in Table 1.

Example 2

Subcloning of plasmid pNM1 to form pNN111

In order to pinpoint the position of the CPG₂ gene and the leader sequence polynucleotide within the 5.9 Md insert, subcloning of various restriction enzyme fragments, into pBR322, was undertaken. A functional CPG₂ gene was shown not to occur on Xhol or Sph1 fragments of tne pNMl insert, but was present on a 3.1 Md Bg111 fragment. This latter fragment was cloned into the BamH1 site of pBR322 to give pNMll (6.0 Md). A further reduction in the size of pNMll was achieved by digesting with Sall and religating the resultant fragment to yield pNMlll. In addition, plasmids in which the smaller 0.95 Md Sall fragment had become inserted in the opposite orientation to the parent plasmid (pNM11) were Fol^-. Taken together these subcloning results indicate that the CPG₂ gene and the leader sequence polynucleotide lie between the Bglll site at 4.14 and the SAIl site at 6.03 on pNMl.

Furthermore, the gene contains a Sph1 (5.17), Sa11 (5.07) and at least one Xhol (4.56 and/or 5.56) site. The restriction enzyme cleavage site map of pNMlll is given in Figure 2.

Example 3

Preparation of recombinant plasmid pNM14. (A plasmid containing both the present leader sequence polynucleotide and the CPG₂ structural gene)

The 3.1 Md Bgl II fragment from Example 2 above was partially digested with Sau3A. These fragments were then cloned into the Bam HI site of pAT153 and transformed into E coli W5445. Of the two _Ap^r Tc^s Fol⁺ colonies obtained, one contained a plasmid which had acquired an extra Sal I and Barn HI site, this was pNM 14. The restriction enzyme cleavage site map of pNM 14 is given in Figure 3. Sequencing of the leader sequence polynucleotide and the CPG₂ structural gene present in pNM 14 gave the nucleotide structure shown in Table 1. DNA sequencing of pNM 14 also revealed that the Sal I - Bam HI fragment was a duplication of a segment of DNA from within the insert (marked ^* on Figure 3) composed of two contiguous Sau 3A fragments.

Example 4 and 5

Preparation of recombinant plasmids pNM 21 and pNM 22 (Plasmids containing both the present leader sequence polynucleotide and the CPG₂ structural gene)

The 3.1 Md Bgl II fragment from Example 2 was cloned into the Bam HI site of pAT 153 and transformed into E coli W 5445. Two Ap^r Tc^s Fol^t colonies were obtained, one containing a plasmid pNM 21 in which the fragment was inserted in the opposite orientation to pNM1 and one containing a plasmid pNM22 in which the fragment was inserted in the same orientation as pNM1. The restriction enzyme cleavage site maps of pNM21 and pNM22 are given in Figures 4 and 5 respectively.

The two plasmids, pNM21 and pNM22 both transformed E.coli to Fol , indicating that a pseudomonad promoter was present on the 3.1Md fragment. However, cells carrying the plasmid pNM21, in which the Bg111 fragment was cloned in the opposite orientation to pNMl, exhibited more rapid growth with folic acid as the sole carbon source. This difference was clearly visible on agar medium, where colonies developed concentric yellow 'halos' of precipitated pteroic acid, the insoluble product of folate hydrolysis.

Confirmation that pNM21 gave enhanced expression of CPG₂ over pNM22, was obtained by assaying enzyme production during batch growth of cells containing either plasmid. (The cells were grown in complex medium supplemented with 1% (w/v) glucose and where appropriate 0.05% (w/v) folic acid. The generation time was 56-66 min. The culture was sampled at hourly intervals and whole cells were disrupted by sonication. Enzyme activity was determined in the centrifugal extract). Results are given in Table 2.

The expression of CPG from the plasmids pNM22 and pNM1 was 2.5 units/litre of culture, representing 0.005% soluble protein. In contrast, expression from pNM21 was 3000-3500 units/litre of culture, which represented 4.7% soluble protein. As the cloned gene is inserted into the BamHI site of pAT153, the observed higher expression of pNM21 is almost certainly due to transcriptional read through from the Tc promoter. The low expression of CPG₂ carried on plasmids pNM1 and pNM22 is consistent with the view that Pseudomonas promoters function poorly in E.coli. It is also apparent from Table 2 that in the presence of folate there is a two-fold increase in the specific activity of enzyme measured in cell sonicates. This phenomenon has been observed in all experiments, but does not seem to be associated with classical induction of the CPG₂ gene, as overall enzyme yield in the presence or absence of folate remains at about 3000 u/litre culture. It in fact reflects a consistent depression in the soluble protein levels measured in sonicates from cells grown in the presence of folate. There is no obvious difference in growth rate of cells grown with folate and the reasons for this result are not clear.

Expression of the cloned gene in Ps.putida

The observation that the CPG₂ gene was expressed in E.coli regardless of the orientation of the gene within the vector suggested that the promoter region of the CPG₂ gene had been cloned with the structural gene and the leader sequence polynucleotide. The low expression of CPG₂ within E.coli from its natural promoter (pNM1, pNM22, pNM111) confirmed other findings that Pseudomonas promoters are poorly recognised by E.coli RNA polymerases. It would be expected that if the gene was introduced back into a pseudomonad cellular environment, then improved expression from the Pseudomonas promoter should result. The 3.1 Md BgIII fragment was subcloned into the Pseudomonas cloning vector pKT230 at its single BamBI site. Two plasmids were obtained, pNM31 and pNM32 representing the two possible orientations of the cloned gene. These plasmids were transformed into Ps.putida 2440 by the method of Bagdasarian and Timmis. Pseudomonad cells carrying both plasmids were cultured in minimal salts medium and enzyme production monitored.

Yields of 500-1000 units/litre of culture were obtained regardless of gene orientation within the plasmid. Specific activity of the enzyme in cell sonicates was 1.5 to 4.0 U/mg protein representing 0.3 to 0.7% soluble protein (compared with < 0.05% soluble protein in the donor strain RS-16). This result strongly indicates that the CPG₂ promoter is present and operating in a pseudomonad background. When the same plasmids were transformed into E.coli W5445 12-40 Units/litre were found at specific activity<0.07 U/mg (<0.01% soluble protein).

Periplasmic localisation of CPG₂

There is evidence that CPG2 is located in or near the periplasmic space of Pseudomonas strain RS-16. Pteroic acid, the product of CPG hydrolysis of folic acid is extremely insoluble and is found predominantly outside the cell in both liquid and solid media. Exogeneous pteroic acid is also seen in E.coli cultures containing the cloned gene when folic acid is present in the medium. This is clearly demonstrated by the 'halo' of precipitated pteroic acid observed around colonies carrying plasmids in which expression of CPG₂ is from the Tc promoter of pBR322 (eg pNM21).

The localisation of CPG₂ produced by E.coli cells carrying pNM21 was examined by the separation of cellular proteins into cytoplasmic, periplasmic, and whole membrane fractions. As a control, levels of three marker enzymes, alkaline phosphatase (periplasmic), glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic) and NADH.02 oxidoreductase (membrane-bound), were also determined. As can be seen from Table 3 97% of the CPG₂ activity occurs in the periplasm, equivalent to the marker periplasmic enzyme, alkaline phosphatase. This confirms the presence in pNM21 of a leader sequence polynucleotide next to the CPG₂ gene that codes for a signal polypeptide according to this in vention that promotes the secretion of CPG₂ from the cytoplasm into the periplasmic space.

Carboxypeptidase G₂ synthesised in E.coli

The specific activity of CPG₂ in crude cell extracts of cells carrying pNM21 was 50-fold higher than equivalent extracts from Pseudomonas strain RS-16. To determine whether the cloned gene= product in E.coli had the same properties as CPG2 from the pseudomonad, enzyme was purified from E.coli carrying pNM21. The specific activity of purified CPG₂ (single band SDS-PAGE) was 535 U/mg of protein, which compares to 550 U/mg of protein from the pseudomonad. CPG₂ purified from E.coli clone pNM21 co-chromatographed with CPG₂ from Pseudomonas strain RS-16 at a sub-unit molecular weight value of 42,000 daltons. Km vallues using methotrexate as substrate were 7.4 x 10^-6M and 8.0 x 10^-6M respectively. In addition, antiserum raised against the Pseudomonas enzyme indicated immunological identity between the E.coli and Pseudomonas CPG₂, as a confluent precipitation line was formed on Ouchterlony double diffusion analysis.

Example 6

Preparation of a recombinant plasmid containing both the present leader sequence polynucleotide and the B-Galactosidase structural gene

Plasmid pNM14 (Example 3) was treated with Sau 3A (GATC) and the fragments were cloned into the Bam HI site of M13 mp7 template DNA (single stranded DNA (Step A of Figure 6). The product carrying a 318bp Sau 3A fragment coding for the present signal polypeptide and the first 22 amino acids of CPG₂ (nucleotide sequence of this fragment shown in Table 4) was selected and made double stranded. The DNA coding for the signal polypeptide (and the first part of CPG₂) was then excised as an Eco RI fragment. This Eco RI fragment was then cloned into the promoter cloning vector E.coli pMC1403 (M.J. Casadaban et al, J Bacteriol, 1980, 143, 971), which carries only the structural gene (lac Z) for B-galactosidase (ie no promoter and no ATG start codon) (Steps B and C of Figure 6). Plasmids were obtained in which the Eco RI fragment had inserted in both orientations, however, only those in which fusion of the CPG₂ sequence to the B-galactosidase sequence had occurred (i) yielded a 0.34 Kb fragment upon digestion with BamHI; (ii) enabled the host cell to hydrolyse the colourless lactose analogue, BCIG, and impart a blue colouration to colonies. The 0.34 Kb BamHI fragment has been recloned into M13mp7 and sequenced to confirm that fusion has occurred. The 'precursor' fusion produced will consist of the signal peptide, the first 22 amino acids of CPG₂, 6 amino acids derived from the M13mp7 and pMC1403linker units, and B-galactosidase from its 8th amino acid onward.

Localisation experiments have been performed on cells carrying a plasmid coding for the 'fusion gene' where the cellular proteins have been fractionated into periplasmic, cytoplasmic and membrane fractions. In these experiments an organism (E,coli MC 1061) which is deleted for the lac Z gene was grown in phosphate medium (H.C. Neu et al, J Biol Chem, 1964, 240, 3685) and periplasmic enzymes were released from the harvested cells by conversion to spheroplasts. Separation of soluble proteins (cytoplasmic) from particulate proteins (membrane band) was achieved by soniPating the harvested spheroplasts and subsequent centrifugation at 100,000g for 1hr, to sediment the cell membrane (T.J.Silhary et al, Proc Natl Acad Sci USA, 1976, 73, 3423).

The results given in Table 5 demonstrate the presence of 50% of the B-galactosidase activity in the periplasmic space. This result is in direct contrast to similar work involving fusion of other periplasmic protein signal sequences to B-galactosidase, where the fusion proteins are not exported, but become jammed in the membrane (P.J. Bassfordet al, J Bacteriol, 1979, 139, 19 and S D Emr et al, J Cell, Biol, 1980, 86, 701).

NB. This fragment carries the leader sequence coding for the signal polypeptide, a part of the CPG₂ structural gene coding for the first 22 amino acids of the protein, the ATG start codon, the CPG₂ ribosome binding site (AGGA) and other components of the CPG₂ promoter region.

Example 7

Preparation of a recombinant plasmid, containing both the present leader sequence polynucleotide and the CPG₂ structural gene, able to replicate in E.coli and S.cerevisiae

A 2.03 kilobase BamHI fragment coding for the present signal polypeptide and the entire CPG₂ molecule was cloned in both orientations into the BamHI site of an E. coli/S. cerevisiae shuttle vector pROG5 (R.F. Sherwood and R.K. Gibson, The Molecular Biology of Yeast, 1979, Cold Spring Harbor Publications) to give plasmids pLEC3 and pLEC4 (Figure 7). These plasmids were transformed into S. cerevisiae strain LL20 by the lithium acetate induced transformation method described by Ito et al., J. Bact., 1983, 153, 163. Yields equivalent to 10-20 units/litre of culture volume were obtained regardless of gene orientation within the plasmid. Specific activity of the enzyme in total cell extracts was 0.2-0.3u/mg protein representing 0.005% soluble protein. This level of expression from the pseudomonad promotor in a yeast background is similar to the level found when the gene was reading from its own promotor in E.coli ( 0.01% soluble protein).

Localisation experiments have been performed on yeast cells carrying the above plasmids by sphaeroplasting the cells using standard techniques described by J.B.D. Beggs, Nature, 1978, 275, 105. Periplasmic enzymes, localised outside of the cell membrane, were released when the cell wall was removed. The osmotic stabiliser (1.2M sorbitol) was then replaced by 0.1M Tris-HC1 buffer, pH 7.3 containing 0.2mM ZnCl₂ to lyse the sphaeroplasts and the whole centrifuged at 100,000 x g for 1 hour to separate proteins in the soluble cytoplasmic fraction from membrane bound proteins. The results in Table 6 demonstrate the presence of 64% of the CPG₂ activity in the periplasmic fraction and a further 16% associated with the cell membrane fraction.