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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
  • B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• the present invention relates to a novel process for the preparation of recombinant low density lipoprotein associated phospholipase A2 (LDL-PLA2) and to 5 new recombinant DNAs that direct the expression of LDL-PLA2 that facilitates said preparation.
• LDL-PLA2 low density lipoprotein associated phospholipase A2
• LDL-PLA2 Low density lipoprotein associated phospholipase A2
• LDL-PLA2 is a serine dependent phospholipase which catalyses the hydrolysis of phospholipids with short
• LDL-PLA2 can hydrolyse phospholipids with acyl chains of up to 9 carbon atoms in length at the sn-2 position (Stremler, KE et al (1989) J. Biol. Chem. 264, 5331-5334). Also, LDL-PLA 2
• 25 phospholipids with unsaturated sn-2 acyl substituents is known to generate truncated acyl side chains of the form required by LDL-PLA2 to be substrates (Patel, KD et al (1992) J.
• LDL-PLA2 Due to the ability of LDL-PLA2 to hydrolise both phospholipids with short chain sn-2 substituents, often arising from oxidative cleavage of longer chain sn-2 substituents, and PAF, there has been much interest in the role of this enzyme as both a pro-inflammatory enzyme and an anti-inflammatory enzyme depending on the precise in vivo role adopted (Tew, DG et al (1996) Arterioscler. Thromb. Vase. Biol. 16, 591-599). Although LDL-PLA2 can be obtained conveniently from human plasma, it is not a particularly abundant enzyme (Tew, DG et al (1996) Arterioscler. Thromb. Vase. Biol.
• the invention provides a process for purifying a recombinant LDL-PLA2 polypeptide, comprising the steps: a) applying a cell extract, supernatant or solution containing a recombinant LDL- PLA2 polypeptide, to a zinc chelating column; b) applying the eluate from (a) to a Blue Sepharose column; and c) applying the eluate from (b) to a Q Sepharose column.
• the present invention provides a process which additionally comprises the prior steps of: i) constructing a recombinant vector comprising a cDNA encoding a histidine tagged LDL-PLA2 polypeptide, ii) expressing the polypeptide encoded by the recombinant vector constructed in (a) in a suitable host; iii) isolating the polypeptide from the harvest medium or cell lysate; iv) purifying the polypeptide using a metal matrix affinity column, preferably a nickle column; and v) removing the histidine tag by protease cleavage.
• Said prior steps (i) to (v) are optional steps that may be be carried out to prepare a solution comprising a recombinant LDL-PLA2 polypeptide at a higher degree of purity.
• steps (i) to (v) may also be carried out independently of the aformentioned purification steps (a) to (c). Under these circumstances the LDL-PLA2 polypeptide so prepared may, if required, be subjected to further processing steps, including further purification steps well known in the art.
• the recombinant vector of step (i) directs the expression of a histidine tagged LDL-PLA2 polypeptide of formula (I):
• His refers to the naturally occurring amino acid histidine
• LDL-PLA2 refers to a polypeptide comprising a polypeptide selected from the group consisting of: a) a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:l; b) a polypeptide having the sequence of SEQ ID NO:l; c) a polypeptide encoded by a polynucleotide comprising a polynucleotide having at least 95% identity to the polynucleotide of SEQ ID NO:2; and d) a polypeptide encoded by the polynucleotide sequence of SEQ ID NO:2; or an active fragment of a polypeptide of (a) to (d);
• X and Y are protease cleavage sites; n and p, which may be the same or different, are 0 tolO; and r and s, which may be the same or different, are 0 or 1.
• n or p is 6, thereby providing a "hexa- histidine" (hexa-his) tagged LDL-PLA2 polypeptide, the tag being located at the N- terminal end (when n is 6) or at the C-terminal end (when p is 6) of the LDL-PLA2 polypeptide, or fragment thereof.
• n is 6 and p is 0, providing an LDL-PLA2 polypeptide with a hexa-his tag at the N-terminal end.
• X and Y are endopeptidase cleavage sites, to facilitate removal of the polyhistidine tag by endopeptidase cleavage. More preferably, X and Y are enterokinase cleavage sites.
• one of r and s is 1 and the other is 0. More preferably, r is 1 and s is
• the LDL-PLA2 polypeptides of the present invention are expressed from polynucleotides which may comprise the coding sequence for the mature polypeptide by itself, for instance the nucleotide sequence of SEQ ID NO:2; or a sequence other than the one contained in SEQ ID NO:2, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:l; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
• the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
• Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression vectors. Accordingly, in a further aspect, the present invention relates to expression vectors which comprise a polynucleotide or polynucleotides of the present invention.
• expression vectors can be used in the process of the invention, for instance, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retrovirases, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
• the expression vectors may contain control regions that regulate as well as engender expression. Generally, any vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
• the appropriate nucleotide sequence may be inserted into an expression vector by any of a variety of well- known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989).
• Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
• the recombinant vector is a baculovirus vector.
• the recombinant vector when expressing hexa-his tagged LDL-PLA2, the recombinant vector comprises the N-terminal hexa-his tag vector, pBluebacHis2B (available from InVitrogen). It will be appreciated that cell-free translation systems may also be employed to produce the LDL-PLA2 polypeptide of the invention using RNAs derived from said recombinant vectors.
• the present invention relates to a host cell transformed with a recombinant vector of the present invention.
• host cells include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyees and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergill s cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
• the insect cells Spodoptera Sf9 cells are used as host cells.
• recombinant vectors into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al., (supra). Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
• the present invention provides LDL-PLA2 obtained by a process hereinbefore described.
• Detagged LDL-PLA2 ie. with the his tag removed, although having slight differences in Km and __c at values, has an activity profile very similar to the LDL-PLA2 protein isolated from natural sources and is isolated as a single molecular species.
• the LDL-PLA2 obtained by the process of the present invention may be used in structural and mechanistic studies which will contribute to the elucidation of the activity of the enzyme and also provide insights into the activity of the enzyme in- vivo.
• the LDL-PLA2 enzyme produced and purified using the vectors and methods of the invention can be used in screens to identify compounds that interact with the enzyme, either by inhibition or by activation of the enzyme's activity.
• the LDL-PLA2 enzyme of the invention may be used to raise monoclonal or polyclonal antibodies for use in applications such as diagnostics, for example.
• LDL-PLA2 polypeptide is a polypeptide comprising a polypeptide selected from the group consisting of: a) a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:l; b) a polypeptide having the sequence of SEQ ID NO:l; c) a polypeptide encoded by a polynucleotide comprising a polynucleotide having at least 95% identity to the polynucleotide of SEQ ID NO:2; and d) a polypeptide encoded by the polynucleotide sequence of SEQ ID NO:2; or an active fragment of a polypeptide of (a) to (d).
• An active fragment is a polypeptide derived from a polypeptide having at least 95% identity to the polypeptide of SEQ ID NO:l and which demonstrates LDL-PLA2 activity.
• a fragment may be formed by proteolytic cleavage of an isolated LDL-PLA2 polypeptide, or may be synthesised as a polypeptide fragment using recombinant DNA techniques, using methods well known in the art.
• Recombinantly produced LDL-PLA2 active fragements may be synthesised as mature polypeptides that require no further processing or as part of a larger polypeptide which is then processed to release the active fragment.
• Such LDL-PLA2 polypeptides, including fragments thereof may additionally be fused to other proteins or polypeptides, such as leader or secretory polypeptide sequences or pre-, pro- or prepro- polypepeptide sequences.
• Example 1 Construction of baculovirus expression transfer vectors.
• LDL-PLA 2 LDL-PLA 2 cDNA (WO 95/00649, SmithKline Beecham) was digested with Xbal and
• Xhol and the approximately 1.3kb fragment was ligated into pBacPac 9 vector (Clontech) cut with Xbal and Xhol, using standard techniques (Sambrook, et al. supra).
• LDL-PLA2 cDNA (WO 95/00649, SmithKline Beecham) was digested with HincII-XhoI, and the 1206bp fragment subcloned in-frame with the N- terminal hexa-his tag of pBluebacHis2B (InVitrogen). To facilitate this, pBluebacHis2B was restricted with BamHI, the 5' protruding end converted to a blunt end' by using DNA poiymerase large (Klenow) fragment, and then further digesting with Xhol.
• the 1206bp HincII-XhoI LDL-PLA2 fragment was then ligated into the vector fragment using standard techniques (Sambrook, et al. supra).
• This 1206bp fragment of the LDL-PLA2 cDNA encodes a polypeptide which lacks the N-terminal 39 amino acids, including the putative signal peptide.
• enzyme with the first 41 amino acids removed was the form isolated from human plasma (Stafforini D. M., et al, (1997), J. Biol. Chem.272, 17895-17898.).
• the hexa-his tag and enterokinases site were introduced into the LDL-PLA2 polypeptide, as hereinabove described, such that the detagged protein begins with the sequence DPSSRSAAGTMNKIOVL (double underline shows beginning of LDL-PLA 2 polypeptide sequence).
• the Spodoptera frugiperda (Sf9) cell line was purchased from the European Collection of Animal Cell Cultures (ECACC). The cells were routinely maintained as shaker flask cultures grown at concentrations ranging from 0.2 - 2 x 10° cells ml'S shaken between 120-160 rpm and incubated at 27 °C. Cells were routinely grown in JPL-41 (Life Technologies, Inc) containing lx lipid supplement, lx yeast ultrafiltrate and 0.2% Pluronic F-68. All supplements were purchased from Sigma- Aldrich. Foetal bovine serum (5 % v/v) was added to viral stocks for long term storage, to prevent loss of viral titre.
• ECACC European Collection of Animal Cell Cultures
• Cell lysate was prepared by resuspending Sf9 cells comprising the expression vector of Example 1A, from a 1 1 culture in 40 ml of lysis buffer, 50 mM Tris, 10 mM CHAPS, 10 mM imidazole, 0.5 M NaCl, 0.01 mg/ml pepstatin A, 0.01 mg ml aprotinin, 0.01 mg/ml leupeptin, 0.01 mg/ml antipain, 0.05 mg/ml benzamidine, pH 7.5.
• the cells were submitted to 3 rounds of freeze-thaw using liquid nitrogen. DNAse (2 mg) was added to reduce the viscosity of the sample due to released DNA.
• the lysate was centrifuged at 18000 x g for 30 minutes to pellet cell debris.
• the supernatant was loaded onto a pre- equilibrated (50 mM Tris, 10 mM CHAPS, 0.5 M NaCl, pH 7.5) 100 ml zinc chelating Sepharose column and washed with four column volumes of equilibration buffer.
• LDL- PLA2 was eluted with 50 mM Tris, 10 mM CHAPS, 0.5 M NaCl, pH 7.5, 50 mM imidazole. Active fractions were pooled and the pH reduced to 6.0 by dropwise addition of a solution of 1 M MES acid in 10 mM CHAPS.
• the sample was loaded onto a 100 ml Blue Sepharose column which had been pre-equilibrated in 50 mM MES, 10 mM CHAPS, 0.5 M NaCl, 1 mM EDTA, pH 6.0.
• the column was washed with five column volumes of equilibration buffer followed by eight column volumes of 50 mM MOPS, 10 mM CHAPS, 0.5 M NaCl, 1 mM EDTA, pH 7.5.
• LDL-PLA 2 was eluted using five column volumes of 50 mM Tris, 10 mM CHAPS, 2 M NaCl, 1 mM EDTA, pH 8.5.
• Active fractions were pooled, concentrated by ultrafiltration (YM30) and then dialysed at 4 C against 50 mM Tris, 10 mM CHAPS, 1 mM EDTA, pH 8.0. Following dialysis the protein solution was centrifuged (18000 x g) to remove any precipitates. The supernatant was loaded onto a high performance Q Sepharose column (8 ml, HR 10/10) which had been pre-equilibrated with 50 mM Tris, 10 mM CHAPS, 1 mM EDTA, pH 8.0. The column was washed with equilibration buffer until the absorbance at 280 nm reached a steady baseline.
• LDL-PLA2 was eluted with a linear gradient from 0-1.0 M NaCl over 15 column volumes. The activity eluted at around 0.4 M salt. Active fractions were pooled, concentrated by ultrafiltration (YM30) and the pure material was stored at 4° C.
• Example 4 Purification of hexa-his tagged LDL-PLA2 Cell lysate was prepared by resuspending Sf9 cells comprising the expression vector of Example IB, from a 1 1 culture in 40 ml of lysis buffer, 50 mM Tris, 10 mM CHAPS, 10 mM imidazole, 0.5 M NaCl, 0.01 mg/ml pepstatin A, 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin, 0.01 mg/ml antipain, 0.05 mg ml benzamidine, pH 7.5. The cells were submitted to 3 rounds of freeze-thaw using liquid nitrogen. DNAse (2 mg) was added to reduce the viscosity of the sample due to released DNA. The lysate was centrifuged at
• Active fractions were pooled, concentrated (Amicon YM30) and stored at 4°C.
• the hexa-his tag was removed using recombinant enterokinase according to the manufacturer's protocol (R&D Systems). Briefly, 0.05 mg of pure LDL- PLA2 was incubated with one unit of enterokinase, in the presence of cleavage buffer (20 mM Tris, 50 mM NaCl, 2 mM CaCl2, pH 7.4) for 16 hours at room temperature.
• LDL-PLA2 was purified further by loading onto the nickel NTA column used above. The detagged LDL- PLA2 eluted in the void volume. The active protein was stored at 4°C.
• LDL-PLA2 substrate DNGP l-decanoyl-2-(4- nitrophenylglutaryl) phosphatidylcholine
• the synthetic LDL-PLA2 substrate DNGP was prepared as a 10 mM stock solution in methanol and diluted into buffer as required.
• LDL-PLA2 was added to 50 ⁇ M DNGP in buffer at 37°C.
• the absorbance increase was followed at 400 nm using either a diode array spectrophotometer (Hewlet-Packard) or a 96 well plate reader (Molecular Devices, Tmax) running in kinetic mode.
• Table 1 summarises the purification of recombinant LDL-PLA2 expressed in the baculovirus infected Sf9 cell system. No figure is shown for the Zn 2+ chelating Sepharose column as the absorbance of the imidazole used to elute LDL-PLA2 from this column overwhelms the protein absorbance at 280 nm. Although the data in Table 1 suggests that this step is unproductive in terms of purification, i.e. a purification factor of 1 is observed, the inclusion of this step was found to be essential to obtaining a good overall purification.
• Blue Sepharose chromatography at step 2 allows rapid adjustment of the pH of the Zn 2+ Chelating Sepharose eluate without the need for dialysis.
• Blue Sepharose chromatography provides the key purification step for recombinant LDL-PLA2 and is easily effected with batchwise washing and elution using a combination of pH and salt concentration steps.
• anion exchange chromatography provides protein of high purity and in good yield. All LDL-PLA2 activity elutes as a single peak. It is noteworthy that the apparent yield on this third and final step is >100%. It is possible that prior to this step lipid components carried through the Zn 2+ Chelating Sepharose and Blue Sepharose chromatographies may act as an inhibitor of this phospholipase. Removal of this inhibitory components on anion exchange would then account for an apparent >100% yield.
• N terminal sequencing was used to confirm both the identity of the purified protein as LDL-PLA2. However, the N terminal sequencing showed several N termini. Careful deconvolution of the sequencing data showed that there were at least three N termini in the purified LDL-PLA2. All three N termini were derived from LDL-PLA2 but none were from the expected full length gene product. These data are shown in Table 2.
• the first step is a NTA-Nickel column. After concentration and dialysis, Blue Sepharose chromatography yields protein of reasonable specific activity and high purity. SDS- PAGE analysis of the purification indicates that after the Blue Sepharose chromatography there is only a single protein present. Detagging using enterokinase followed by a repeat of the NTA-Nickel column yields homogeneous detagged LDL-PLA2 now eluting in the void volume. N terminal sequencing confirmed the authenticity of the N-terminus of the detagged protein.
• the Km value obtained for the detagged LDL-PLA2 is 20 ⁇ M whilst the k ⁇ value is 68 s"l respectively.
• LDL-PLA2 purified from human plasma has a Km of 12 ⁇ M and a kcat of 100 s' 1 (Tew, DG et al (1996) Arterioscler. Thromb. Vase. Biol. 16, 591-599).
• the detagged recombinant form of LDL-PLA2 is strikingly similar to the native enzyme isolated from human plasma.
• the slightly lower Km value may be attributable to the deletion at the N-terminus of the protein.
• the Xbal and Xhol sites used for the subcloning described in Example 1A were provided by the ⁇ Unizap XR vector (Stratagene) vector comprising the LDL-PLA2 cDNA (WO95/00649, SmithKline Beecham) and are therefore not shown on the above sequence.
• the Xhol site is approximately 40bp downstream of the stop codon in the ⁇ Unizap XR vector that was used for cloning the LDL-PLA2 cDNA (WO 95/00649, SmithKline Beecham).

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