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TECHNIQUES
[19]
tail in close proximity to this region in the Group II PLA2. Thus far, however, no functional distinction exists that would support such a view. Conclusions Our understanding of the complex relationship between the structure and the function of proteins remains primitive and rudimentary, despite enormous advances in the technology surrounding structural analysis. We still do not know how the information encoded in the amino acid sequence is translated into folding and function of the protein in question. Indeed, this area of research has been, and continues to be, one of the most challenging in contemporary biochemistry. As was mentioned above, we see Nature's mutagenesis in the dozens of PLA 2 sequences available for examination. Accordingly, we have gained insights as to what parts of the molecule are retained and, therefore, essential for function, be it with respect to calcium binding, esterolysis, protein-protein interaction, enzyme-substrate complexation, or toxicology. Nevertheless, it is clear that we have a long way to go in the process of unraveling the secrets of even so simple a class of enzymes as the PLA2. It is hoped that the present chapter provides a rationale and chemical basis for probing structure-function relationships in the PLA2 that will, in turn, help to answer some of these intriguing questions of more general significance.
[19] C l o n i n g , E x p r e s s i o n , a n d P u r i f i c a t i o n o f P o r c i n e Pancreatic Phospholipase A 2 and Mutants By H. M. VEgHEu and G. H. DE HAAS
Introduction Phospholipase A2 (EC 3.1.1.4) attacks the acyl ester bond at position 2 of 3-sn-phosphoglycerides.l The in oivo importance of phospholipase A 2 (PLA2) is reflected by the fact that this enzyme occurs ubiquitously in nature and that PLA2 activity has been detected in a large number of cell types and cell organeUes. In general their physiological role can be either I L. L. M. van Deenen and G. H. de Haas,
METHODS IN ENZYMOLOGY, VOL. 197
Biochim. Biophys. Acta 70, 538 (1963). Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[19]
RECOMBINANT PANCREATICPHOSPHOLIPASEA2
215
a digestive or a regulatory one. The exracellular PLA2s from mammalian pancreas and snake venom undoubtedly belong to the digestive enzymes whereas the cellular PLAzs have been assigned regulatory functions mainly. 2 The PLA2s are relatively small proteins (14 kDa; about 120 amino acids) and display a very high stability to denaturing conditions, such as high temperature and low pH. The high stability probably is related to the large number (six or seven) of disulfide bridges. The primary structure has been elucidated for about 65 extracellular and 5 cellular PLA2s. A comparison of these sequences 3 (see also this volume [18]) reveals a high degree of homology. In addition, crystallographic data show striking similarities in the three-dimensional structure of PLA2s from bovine and porcine pancreas and enzyme from the venom of C r o t a l u s a t r o x . 4 The sequence similarities and the structural resemblances suggest a general mode of action for all PLA2s. For pancreatic PLAzs a mechanism of action has been proposed 5 in which an Asp-His couple serves as a proton relay system, resembling that of the serine proteases. Contrary to these esterases, however, PLA2 lacks a serine in the active center, and a water molecule is held responsible for the nucleophilic attack. The cellular PLA2s are membrane-associated enzymes which normally occur in a dormant form; their activation is a matter of debate (see [18] in this volume). The pancreatic PLA2s are produced and stored in the pancreas in the form of an inactive precursor, and activation involves the removal by trypsin of an amino-terminal heptapeptide. The occurrence of this natural cleavage site has been the basis for the successful expression of porcine pancreatic PLA2. Cloning of Porcine Pancreatic Prophospholipase A s Cloning of PLA2s from various sources has been achieved using synthetic genes, 6'7 genomic banks 8,9 and cDNA banks, s'1°-12 For the general M. Waite, in "Handbook of Lipid Research" (D. J. Hanahan, ed.), Vol. 5, p. 155. Plenum, New York, 1987. 3 C. J. van den Bergh, A. J. Slotboom, H. M. Verheij, and G. H. de Haas, J. Cell. Biochem. 39, 379 (1989). 4 R. Renetseder, S. Brunie, B. W. Dijkstra, J. Drenth, and P. B. Sigler, J. Biol. Chem. 260, 11627 (1985). 5 H. M. Verheij, J. J. Volwerk, E. H. J. M. Jansen, W. C. Puijk, B. W. Dijkstra, J. Drenth, and G. H. de Haas, Biochemistry 19, 743 (1980). 6 j. p. Noel and M.-D. Tsai, J. Cell. Biochem. 40, 309 (1989). 7 T. Tanaka, S. Kimura, and Y. Ota, Gene 64, 257 (1988). 8 j. j. Seilhamer, T. L. Randall, Y. Miles, and L. K. Johnson, DNA 5, 519 (1986). 9 R. M. Kramer, C. Hession, B. Johansen, G. Hayes, P.McGray, E. P. Chow, R. Tizzard, and R. B. Pepinsky, J. Biol. Chem. 264, 5768 (1989).
216
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[19]
methodology and references, readers are referred to these publications. The banks were screened in most cases with the aid of oligonucleotides or genetic probes and occasionally in an expression library with antibodies against the PLA 2 .12 The eDNA banks of porcine pancreatic PLA 2 were obtained s,l° starting from fresh pancreatic tissue that was frozen immediately in liquid nitrogen. From this tissue RNA and poly(A) ÷ RNA were isolated by established procedures.13,14 After eDNA synthesis, Seilhamer et al. 8 obtained about 1.6 x 105 independent hgtl0 clones per microgram RNA. After synthesis of eDNA by the Gubler and Hoffman procedure, 15,16de Geus et al. 1° obtained, per microgram RNA, about 1.5 × 103 independent clones carrying an insert in the P s t I site of pBR322. These banks were screened with the aid of degenerated synthetic oligonucleotides that were based on the known amino acid sequence. From the eDNA bank from porcine pancreas, a clone containing a 560-base pair cDNA insert was sequenced and was found to cover the complete coding region for the preproPLA 2 .10 The predicted amino acid sequence (Fig. 1) is in full agreement with the one known for porcine proPLA2.17 Expression experiments in eukaryotic cell lines confirmed the functional integrity of the signal peptide sequence, since the proPLA 2 was secreted into the culture medium.18 Expression of (Pro)phospholipase A 2 In general, the expression of cloned eukaryotic proteins, at levels that permit characterization of the recombinant protein with methods requiring substantial amounts of protein, isfar from being straightforward. In particular, posttranslational modifications can cause serious complications. A n example of such a modification is the formation of the disulfide bonds which happen to be abundant in all PLA2s. T w o alternative approaches
10 p. de Geus, C. J. van den Bergh, O. P. Kuipers, H. M. Verheij, W. P. M. Hoekstra, and G. H. de Haas, Nucleic Acids Res. 15, 3743 (1987). 11 O. Ohara, M. Tamaki, E. Nakamura, Y. Tsuruta, Y. Fujii, M. Shin, H. Teraoka, and M. Okamoto, J. Biochem. (Tokyo) 99, 733 (1986). 12 K. Kiichler, M. Gmachl, M. J. Sippl, and G. Kreil, Eur. J. Biochem. 184, 249 (1989). J3 j. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and J. Rutter, Biochemistry 18, 5294 (1979). 14 H. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972). 15 U. Gubler and J. Hoffman, Gene 25, 263 (1983). 16 U. Gubler, this series, Vol. 152, p. 330. t7 W. C. Puijk, H. M. Verheij, and G. H. de Haas, Biochirn. Biophys. Acta 492, 254 (1977). 18 p. de Geus, O. P. Kuipers, M. van den Heuvei, H. M. Verheij, and G. H. de Haas, Chim. (Ottobre), p. 73 (1987).
[19]
RECOMBINANT PANCREATIC PHOSPHOLIPASE
--~oJ~(n~
6or rrr Gcr CAC CAA ccr
A2
217
5O
GAC AGe AGG I m l ~ -15
CTC GTG TTG @CT G ~ Leu Vol Leu Alo Vol
CTG CTC ACA Leu Leu Thr iso TTA Trig CAG TTT CGT AGe ATr, All" Leu Trp Gln Phe Arg Ser Met lle TTC Phe 250 GAC AsP AAA Lys ATC IIe GCT AIo
rrc
Lys
Phe
I00 GIG @GC GCT @CC ~ cAG GAA GGC ATC AGC TCA AG6|GCA Vol GIy Alo Alo Gin Glu GIy lle Ser Ser Arg Alo
AAG TGC @CA ATC CCC GlC AGT CAC CCC TT6 AT n BAT LYe Cys Alo lle Pro GIy Ser HIe Pro Leu Met ASP io 20
200 AAC AAC TAT GGC TGC TAC TGT G6C CTA GGT GGA TCA GGG ACC CCT GTG GAT 6AA CTG Asn Asn Tyr 61y Cys Tyr Cys 61y Leu GIY GIy Ser GIy Tier Pro Vol ASP Glu Leu 3o 40
AGG TGC TGC GAG ACA CAC GAC AAC TGC TAC AGA GAT GCC AAG Arg CYS Cys G]u Thr HIs Asp Ash Cys Tyr Arg AsP Alo Lye 50 35o TTC CTC GTG GAC AAT CCC TAC ACC GAA AGC TAC TCC TAC TCA PtTe keu Vol ASp Asn Pro Tyr Tier GIu Ser Tyr Set Tyr Ser 70 4OO ACC TGC AAC AGC AAA AAC AAT @CT TGT GAG GCC TTC ATC TGT Thr Cys Asn Ser LYs Ash Asn Alo Cys Glu A]o Phe I]e Cys 90 450 GCC ATT TGC TTC TCA AAG GCC CCA TAC AAC AAG GAG CAC AAG A]o lie Cys Phe Ser Lys Alo Pro Tyr Asn Lys Glu His LyS
300 AAC CTG GAC AGC TGT Aen Leu Asp Ser Cys 60
TGT TCT AAC ACT GAG Cys Ser Aen Thr Glu 80
AAC TGT GAC CGA AAT Ash Cye ASP Arg Aen 100 AAC CTG GAC ACC AAG Asn Leu ASD Thr Lys
II0
120
50(9 AAG TAC TGT TAG AGC TAA GTA TCA CCC Lys Tyr Cys " ' * ### 124
FIG. 1. cDNA and deduced protein sequence of porcine pancreatic preproPLA2. The triangle represents the cleavage site for signal peptidase. Roman numerals denote residues of the activation peptide of proPLA 2 , which is cleaved by trypsin ( a r r o w ) .
can be chosen to solve this problem. First, for expression eukaryotic cells can be used which are capable of in vivo disulfide bond formation. Second, a prokaryote like Escherichia coli can be used as expression host to produce (partly) incorrectly folded protein, and subsequent in vitro refolding and oxidation may then give folded protein with the correct disulfide bonds. Reduction and reoxidation has been shown to be an efficient process both for pancreatic (pro)PLAz and for snake venom PLA 2.19,20 Another problem that is often encountered during heterologous expression deals with the instability of the protein of interest due to cellular proteases.
Expression in Escherichia coli During our experiments aimed at the expression in E. coli, it became evident that porcine pancreatic proPLA2 could not be expressed to any significant levels, either directly in the cytoplasm, or after processing into 19G. J. M. van Scharrenburg, G. H. de Haas, and A. J. Slotboom, Hoppe-Seyler'sZ. Physiol. Chem. 361, 571 (1980). 20 S. Tanaka, Y. Takahashi, N. Mohri, H. Kihara, and M. Ohno, J. Biochem. (Tokyo) 96, 1443 (1984).
218
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[19]
the periplasmic space. For this reason we developed a strategy for the expression of PLA2 as a 59-kDa fusion protein which precipitates inside the cell. The approach combines several published methodsl°: (1) purification of insoluble fusion proteins from the cytoplasm, (2) S-sulfonation and subsequent reoxidation and renaturation of recombinant proteins, and (3) site-specific cleavage of the fusion protein with hydroxylamine or trypsin. Hydroxylamine cleavage of the peptide bond beteen Ash and Gly residues, a sequence which is absent in porcine pancreatic PLA 2, was optimized by the insertion of three consecutive Asn-Gly sequences between proPLA2 and the bacterial leader fragment of the fusion protein. For tryptic cleavage the "natural" cleavage site, namely, the Arg-Ala 1 bond, is used. FQr bovine pancreatic PLA2 an expression system has been published which makes use of a preproPLA 2 construct giving rise to the precipitation of the desired protein inside the cell, and a protocol was given for small-scale purification. 6 In our laboratory E. coli K12 strain AB1157 was used originally 1° as the host organism for the pEX-derived expression vector and for vector plasmid pCI857, carrying the temperature-sensitive h repressor. At a later stage this strain was replaced by strain MC 4100, 21 a strongly growing strain with a chromosomal lacZ deletion. More recently the DNA fragment coding for the truncated Cro-lacZ-proPLA2 fusion • protein • 10has been introduced into vector pUEX, 22 which by itself carries the temperature-sensitive h repressor. Although the protein yields are similar, the main advantage is the improved ease of manipulating one plasmid instead of two. To produce fusion protein in shaking bottles, a fresh overnight culture, grown at 30° in the presence of antibiotics, is diluted 5-fold with warm (41 °) medium. Induction is continued for 3 hr, after which the cells are harvested by centrifugation. In a fermentor (New Brunswick Microferm Fermentor Model MF-114, New Brunswick Scientific Co., New Brunswick, N J) the overnight starter culture is used to inoculate a 10-fold volume of medium. Stirring is set at 400 rpm, and aeration is kept at 300 ml/min per liter medium. Growth is continued at 30° until the OD600 is 0.6 (about 2 hr) after which the temperature is shifted in about 20 min to 41 °. The OD6o0increases to about 3.5, and after 3 hr of induction cells are harvested by centrifugation. Cells are broken by sonication after lysozyme treatment, protein aggregates are isolated from cell lysates by a 30-min centrifugation step at 5000 g; and the protein pellet is washed once with 0.1% Triton X-100. Starting from a 10-liter culture, a typical yield is 200-400 mg
n M. J. Casadaban, J. Mol. Biol. 104, 541 (1976). 22 G. M. Bressan and K. K. Stanley, Nucleic Acids Res. 15, 10056 (1987).
[19]
RECOMBINANT PANCREATICPHOSPHOLIPASEA2
~
219
Inclusionbodies Dissolve
Reducti~/._(. "~eduction/rena tur:~i~ GOOH
2H N ~
~ Tryl:)sin
Hydroxylamine/ Renaturation
H2N~ ' ~
PRO-PLA
COOH
*PRO-PLA
H2N. ~ ~ ) COOH PLA
FIG. 2. Schematic representation of the refolding of PLA2 and cleavage to proPLA2 and active PLA2, respectively. The black arrow represents the trypsin cleavage site, ---(3 a free SH group, ------(3---(3---a disulfide bond, and t2 the Asn-Gly linker.
fusion protein, being the major band on sodium dodecyl sulfate (SDS)polyacrylamide gels. The order of subsequent steps is outlined schematically in Fig. 2. The fusion protein is first sulfonated at 2-5 mg/ml, as described by Tannhauser and Sheraga. 23 The S-sulfo protein is precipitated by 5 volumes of a cold 1% (w/v) acetic acid solution. The precipitate is collected by centrifugation, and the pellet is washed several times with water. The pellet originating from a l-liter culture is dissolved in 20 ml of 8 M urea, 100 mM sodium borate (pH 8.5), 20 mM EDTA. After dilution to 80 ml, reduced and oxidized glutathione are added to final concentrations of 2 and I mM, respectively. Renaturation usually reaches a maximal level within 24 hr at room temperature, as measured in an L-dioctanoyllecithin assay) ° The (partly) renatured fusion protein is assayed for its content of active PLA2 by adding a portion to the micellar assay reaction vessel, followed by l0 /zg of trypsin. Within minutes the tryptic activation is complete as judged from the fact that the recorded hydroxide uptake becomes linear with time. 23 T. W. Tannhauser and H. A. Scheraga, Biochemistry 24, 7681 (1985).
220
PHOSPHOLIPASE STRUCTURE--FUNCTION TECHNIQUES
[19]
After dialysis, preparative tryptic digestion of the renatured fusion protein is done in a buffer containing 20 mM Tris (pH 8) and 5 mM CaCI 2. Trypsin (5/zg/mg fusion protein) is added in the beginning and after I hr. Normally, after 2-3 hr of incubation at room temperature the PLA2 activity reaches a maximum. The mixture is acidified to pH 4.5, dialyzed against 5 mM sodium acetate, pH 4.8, and centrifuged to remove insoluble peptides. Occasionally the pellet may contain residual PLA 2 activity. If so, the pellet is redissolved at pH 8, acidified to pH 4.8, and centrifuged. The combined supernatants are applied to a carboxymethyl-cellulose column (20 ml for 100.mg fusion protein) equilibrated at pH 4.8 with 5 mM sodium acetate. The PLA2-containing fractions are further purified by two consecutive columns: a carboxymethyl-cellulose column at pH 6 and a diethylaminoethyl-cellulose column at pH 7.5-8.5, depending on the isoelectric point of the mutant PLA 2. In Fig. 3 the elution patterns for a Y69F mutant PLA2 are depicted. Starting from a 10-liter culture, the yield of pure PLA2 is 40-90 mg, reflecting a nearly quantitative conversion of 200-400 mg of the 59-kDa fusion protein. ProPLA2 is isolated from the fusion protein by cleavage with hydroxylamine. First, the inclusion bodies are solubilized under reducing conditions, at 5 mg/ml in 6 M guanidine, 50 mM Tris (pH 8), 1 mM EDTA and 5 mM 2-mercaptoethanol, for 1-3 hr under N2. The mixture is then centrifuged to remove insoluble particles, and the supernatant is diluted 10-fold with cold 0.1% acetic acid. The precipitated fusion protein is collected by centrifugation, and the pellet is washed 3 times with dilute acid. The pellet is dissolved, at a protein concentration of 5 mg/ml, in 6 M guanidine, 2 M hydroxylamine, adjusted to pH 8.5 with LiOH. After a 3 hr incubation at 45 °, the reaction is stopped by the addition of 0.1 volume acetic acid followed by dialysis against 1% acetic acid. Insoluble material is removed by centrifugation, and the soluble fraction is adjusted to pH 8 with NaOH. Additions are made to the following final concentrations: guanidine (1.5 M), Tris-HC1 (50 mM, pH 8.0), Ca 2÷ (I0 mM), reduced glutathione (2 mM), and oxidized glutathione (1 mM). Following renaturation and dialysis, proPLA 2 is purified as described above for the active enzyme. From a 10-liter culture about 15 mg of pure proPLA 2 is obtained.
Expression in Yeast
Whereas expression of PLA2 in E. coli does not lead to the production of active (pro)PLA2 because no or incorrect formation of disulfide bonds occurs in the cytoplasm, the yeast Saccharornyces cerevisiae is capable of both in vivo disulfide bridge formation and efficient secretion of heterologous proteins. Therefore, this yeast has been used as a host system for
[19]
RECOMBINANT PANCREATIC PHOSPHOLIPASE A 2
/ z
221
p.4.8 CM
0 10 20 30 40 50 60 70
ilC I1: I-
CM pH 6.0
n-
O
co
d 6
0
A
10
20
30
I I DEAE pH 8.0
0 10 20 30 FRACTION NUMBER
FIG. 3. Elution profiles of Y69F mutant porcine pancreatic PLA 2 after tryptic digestion of fusion protein. The recorded profiles were obtained starting from a 10-liter culture. Phospholipase A 2 activity is indicated by hatched areas. CM, pH 4.8, and CM, pH 6.0: carboxymethyl-cellulose columns run at pH 4.8 and 6.0, respectively; DEAE, pH 8.0: diethylaminoethyl-cellulose column run at pH 8.0. Proteins were eluted from the columns with linear gradients of NaCl of 0-0.5, 0-0.4, and 0-0.2 M, respectively. For details, see text.
the expression and secretion of several mammalian enzymes. Both bovine 7 and porcine 24-26 (pro)PLA2 have been expressed and purified from this organism. In our laboratory constitutive expression of proPLA 2 in S. cerevisiae was obtained after fusing the proPLA2 to the prepro sequence of the yeast a-mating factor. 24On secretion, the fusion protein was cleaved by the KEX2 protease, yielding a 140-amino acid long precursor form of the PLA2. This protein, present in concentrations of about 600/zg/liter 24 C. J. van den Bergh, A. C. A. P. A. Bekkers, P. de Geus, H. M. Verheij, and G. H. de Haas, Fur. J. Biochem. 140, 241 (1987). 25 C. J. van den Bergh, Ph.D. Thesis, University of Utrecht (1989). 26 A. C. A. P. A. Bekkers, P. A. Franken, C. J. van den Bergh, J. Verbakel, H. M. Verheij, and G. H. de Haas, submitted for publication.
222
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[19]
culture supernatant, was easily purified by ion-exchange chromatography. In later experiments 25 we have replaced the strong constitutive promoter of the yeast a-mating factor by the galactose-inducible G A L 7 promoter. The construct furthermore contains the invertase signal sequence preceding the a-mating factor prosequence-proPLA 2 fusion. 26 Cells harboring this construct target proPLA2 into the culture medium to a level between 2.5 and 6 mg/liter. A similar level of production has been observed by Tanaka et a l . 7 for the expression/secretion of bovine pancreatic proPLA 2 by yeast under control of the P H 0 5 promoter. Whereas in our laboratory only proPLA2 was excreted, Tanaka et al. obtained a mixture of precursor and active PLA2. It is not clear whether this partial activation can be explained by the use of different expression constructs or whether it is strain dependent. Purification of yeast proPLA2 from fermentation supernatants of 10 liters or more can be accomplished with the use of ion-exchange chromatography, 24 thereby circumventing the use of Sephadex columns at an early stage of the purification method as was done by Tanaka et al. 7 After removal of the cells by centrifugation, the culture medium is acidified to pH 3.5, and 15 ml of preswollen SP-Sephadex C-25 is added per liter of medium. After gentle stirring for several hours at 4 °, the beads are collected and washed first with 10 mM formate buffer, pH 3.5, and then with 10 mM acetate buffer, pH 5.0. In this step proPLA2 binds nearly quantitatively to the ion-exchange resin, whereas most colored material remains unbound. I
I
I
I
I
I
I
-
.
0.4
:[
0.2~
.z
0 10
20 30 40 FRACTION NUMBER
50
FIG. 4. Purification of yeast proPLA2 by SP-Sephadex chromatography. The supernatant from 10 liters of culture broth of the strain harboring the GAL7 construct encoding native porcine pancreatic PLA2 (see text) was acidified to pH 3.5 and bound to 150 ml SP-Sephadex. After several wash steps the beads were loaded on top of a column containing 250 ml fresh SP-Sephadex. For details, see text.
[20]
ANTIBODIES TO PHOSPHOLIPASES
A2
223
The SP-Sephadex beads are then packed into a column on top of a fresh layer of SP-Sephadex C-25. The column is eluted with a salt gradient from 0 to 0.5 M NaCI. The proPLA2 peak elutes after the majority of the strongly absorbing colored material which forms the large peak seen in Fig. 4. At this stage the proPLA 2 is the major band on SDS-polyacrylamide gel electrophoresis. After dialysis, the proPLA2-containing fractions are further purified on a carboxymethyl-cellulose column at pH 6.0 in 5 mM acetate buffer. The resulting preparation is desalted and freed from traces of colored material on a Sephadex G-50 fine column in 10 mM acetic acid. Yeast proPLA 2 has the same turnover number for monomeric substrates as native proPLA 2 , although its activation peptide in the a-mating factor and in the G A L 7 constructs is 9 amino acids longer than in native proPLA2. Apparently the extra amino acids do not hinder the entrance of substrate to the active site; they are probably fully exposed to the solvent and do not form contacts with any other part of the enzyme. This is also evident from the fact that yeast proPLA2 is as rapidly cleaved by trypsin as is native proPLA 2 . The resulting yeast PLA2 is indistinguishable from authentic pancreatic PLA2, showing that a protein with as many as seven disulfide bridges per 124 amino acid residues is correctly processed by the yeast secretory apparatus. Yeast, rather than E. coli, may therefore be the organism of choice to express PLA 2 mutants with impaired folding properties.
[20] P r e p a r a t i o n o f A n t i b o d i e s to P h o s p h o l i p a s e s A 2 B y MAKOTO M U R A K A M I , KIYOSHI TAKAYAMA, MASATO U M E D A , ICHIRO K U D O ,
and KEIZO INOUE
Introduction P h o s p h o l i p a s e s A 2 play important roles in several biological phenomena such as membrane phospholipid turnover, production of lipid mediators (eicosanoids, platelet-activating factor, etc.),~ and the process of inflammation. 2,3 Phospholipases A 2 a r e classified into two groups in terms z H. van den Bosch, Biochim. Biophys. Acta 6114, 191 (1980). 2 p. Vadas and W. Pruzanski, Lab. Invest. 55, 391 (1986). 3 p. Vadas, S. Wasi, H. Z. Movat, and J. B. Hay, Nature (London) 293, 583 (1981).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
PHOSPHOLIPASE STRUCTURE-FUNCTION
TECHNIQUES
[19]
tail in close proximity to this region in the Group II PLA2. Thus far, however, no functional distinction exists that would support such a view. Conclusions Our understanding of the complex relationship between the structure and the function of proteins remains primitive and rudimentary, despite enormous advances in the technology surrounding structural analysis. We still do not know how the information encoded in the amino acid sequence is translated into folding and function of the protein in question. Indeed, this area of research has been, and continues to be, one of the most challenging in contemporary biochemistry. As was mentioned above, we see Nature's mutagenesis in the dozens of PLA 2 sequences available for examination. Accordingly, we have gained insights as to what parts of the molecule are retained and, therefore, essential for function, be it with respect to calcium binding, esterolysis, protein-protein interaction, enzyme-substrate complexation, or toxicology. Nevertheless, it is clear that we have a long way to go in the process of unraveling the secrets of even so simple a class of enzymes as the PLA2. It is hoped that the present chapter provides a rationale and chemical basis for probing structure-function relationships in the PLA2 that will, in turn, help to answer some of these intriguing questions of more general significance.
[19] C l o n i n g , E x p r e s s i o n , a n d P u r i f i c a t i o n o f P o r c i n e Pancreatic Phospholipase A 2 and Mutants By H. M. VEgHEu and G. H. DE HAAS
Introduction Phospholipase A2 (EC 3.1.1.4) attacks the acyl ester bond at position 2 of 3-sn-phosphoglycerides.l The in oivo importance of phospholipase A 2 (PLA2) is reflected by the fact that this enzyme occurs ubiquitously in nature and that PLA2 activity has been detected in a large number of cell types and cell organeUes. In general their physiological role can be either I L. L. M. van Deenen and G. H. de Haas,
METHODS IN ENZYMOLOGY, VOL. 197
Biochim. Biophys. Acta 70, 538 (1963). Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[19]
RECOMBINANT PANCREATICPHOSPHOLIPASEA2
215
a digestive or a regulatory one. The exracellular PLA2s from mammalian pancreas and snake venom undoubtedly belong to the digestive enzymes whereas the cellular PLAzs have been assigned regulatory functions mainly. 2 The PLA2s are relatively small proteins (14 kDa; about 120 amino acids) and display a very high stability to denaturing conditions, such as high temperature and low pH. The high stability probably is related to the large number (six or seven) of disulfide bridges. The primary structure has been elucidated for about 65 extracellular and 5 cellular PLA2s. A comparison of these sequences 3 (see also this volume [18]) reveals a high degree of homology. In addition, crystallographic data show striking similarities in the three-dimensional structure of PLA2s from bovine and porcine pancreas and enzyme from the venom of C r o t a l u s a t r o x . 4 The sequence similarities and the structural resemblances suggest a general mode of action for all PLA2s. For pancreatic PLAzs a mechanism of action has been proposed 5 in which an Asp-His couple serves as a proton relay system, resembling that of the serine proteases. Contrary to these esterases, however, PLA2 lacks a serine in the active center, and a water molecule is held responsible for the nucleophilic attack. The cellular PLA2s are membrane-associated enzymes which normally occur in a dormant form; their activation is a matter of debate (see [18] in this volume). The pancreatic PLA2s are produced and stored in the pancreas in the form of an inactive precursor, and activation involves the removal by trypsin of an amino-terminal heptapeptide. The occurrence of this natural cleavage site has been the basis for the successful expression of porcine pancreatic PLA2. Cloning of Porcine Pancreatic Prophospholipase A s Cloning of PLA2s from various sources has been achieved using synthetic genes, 6'7 genomic banks 8,9 and cDNA banks, s'1°-12 For the general M. Waite, in "Handbook of Lipid Research" (D. J. Hanahan, ed.), Vol. 5, p. 155. Plenum, New York, 1987. 3 C. J. van den Bergh, A. J. Slotboom, H. M. Verheij, and G. H. de Haas, J. Cell. Biochem. 39, 379 (1989). 4 R. Renetseder, S. Brunie, B. W. Dijkstra, J. Drenth, and P. B. Sigler, J. Biol. Chem. 260, 11627 (1985). 5 H. M. Verheij, J. J. Volwerk, E. H. J. M. Jansen, W. C. Puijk, B. W. Dijkstra, J. Drenth, and G. H. de Haas, Biochemistry 19, 743 (1980). 6 j. p. Noel and M.-D. Tsai, J. Cell. Biochem. 40, 309 (1989). 7 T. Tanaka, S. Kimura, and Y. Ota, Gene 64, 257 (1988). 8 j. j. Seilhamer, T. L. Randall, Y. Miles, and L. K. Johnson, DNA 5, 519 (1986). 9 R. M. Kramer, C. Hession, B. Johansen, G. Hayes, P.McGray, E. P. Chow, R. Tizzard, and R. B. Pepinsky, J. Biol. Chem. 264, 5768 (1989).
216
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[19]
methodology and references, readers are referred to these publications. The banks were screened in most cases with the aid of oligonucleotides or genetic probes and occasionally in an expression library with antibodies against the PLA 2 .12 The eDNA banks of porcine pancreatic PLA 2 were obtained s,l° starting from fresh pancreatic tissue that was frozen immediately in liquid nitrogen. From this tissue RNA and poly(A) ÷ RNA were isolated by established procedures.13,14 After eDNA synthesis, Seilhamer et al. 8 obtained about 1.6 x 105 independent hgtl0 clones per microgram RNA. After synthesis of eDNA by the Gubler and Hoffman procedure, 15,16de Geus et al. 1° obtained, per microgram RNA, about 1.5 × 103 independent clones carrying an insert in the P s t I site of pBR322. These banks were screened with the aid of degenerated synthetic oligonucleotides that were based on the known amino acid sequence. From the eDNA bank from porcine pancreas, a clone containing a 560-base pair cDNA insert was sequenced and was found to cover the complete coding region for the preproPLA 2 .10 The predicted amino acid sequence (Fig. 1) is in full agreement with the one known for porcine proPLA2.17 Expression experiments in eukaryotic cell lines confirmed the functional integrity of the signal peptide sequence, since the proPLA 2 was secreted into the culture medium.18 Expression of (Pro)phospholipase A 2 In general, the expression of cloned eukaryotic proteins, at levels that permit characterization of the recombinant protein with methods requiring substantial amounts of protein, isfar from being straightforward. In particular, posttranslational modifications can cause serious complications. A n example of such a modification is the formation of the disulfide bonds which happen to be abundant in all PLA2s. T w o alternative approaches
10 p. de Geus, C. J. van den Bergh, O. P. Kuipers, H. M. Verheij, W. P. M. Hoekstra, and G. H. de Haas, Nucleic Acids Res. 15, 3743 (1987). 11 O. Ohara, M. Tamaki, E. Nakamura, Y. Tsuruta, Y. Fujii, M. Shin, H. Teraoka, and M. Okamoto, J. Biochem. (Tokyo) 99, 733 (1986). 12 K. Kiichler, M. Gmachl, M. J. Sippl, and G. Kreil, Eur. J. Biochem. 184, 249 (1989). J3 j. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and J. Rutter, Biochemistry 18, 5294 (1979). 14 H. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972). 15 U. Gubler and J. Hoffman, Gene 25, 263 (1983). 16 U. Gubler, this series, Vol. 152, p. 330. t7 W. C. Puijk, H. M. Verheij, and G. H. de Haas, Biochirn. Biophys. Acta 492, 254 (1977). 18 p. de Geus, O. P. Kuipers, M. van den Heuvei, H. M. Verheij, and G. H. de Haas, Chim. (Ottobre), p. 73 (1987).
[19]
RECOMBINANT PANCREATIC PHOSPHOLIPASE
--~oJ~(n~
6or rrr Gcr CAC CAA ccr
A2
217
5O
GAC AGe AGG I m l ~ -15
CTC GTG TTG @CT G ~ Leu Vol Leu Alo Vol
CTG CTC ACA Leu Leu Thr iso TTA Trig CAG TTT CGT AGe ATr, All" Leu Trp Gln Phe Arg Ser Met lle TTC Phe 250 GAC AsP AAA Lys ATC IIe GCT AIo
rrc
Lys
Phe
I00 GIG @GC GCT @CC ~ cAG GAA GGC ATC AGC TCA AG6|GCA Vol GIy Alo Alo Gin Glu GIy lle Ser Ser Arg Alo
AAG TGC @CA ATC CCC GlC AGT CAC CCC TT6 AT n BAT LYe Cys Alo lle Pro GIy Ser HIe Pro Leu Met ASP io 20
200 AAC AAC TAT GGC TGC TAC TGT G6C CTA GGT GGA TCA GGG ACC CCT GTG GAT 6AA CTG Asn Asn Tyr 61y Cys Tyr Cys 61y Leu GIY GIy Ser GIy Tier Pro Vol ASP Glu Leu 3o 40
AGG TGC TGC GAG ACA CAC GAC AAC TGC TAC AGA GAT GCC AAG Arg CYS Cys G]u Thr HIs Asp Ash Cys Tyr Arg AsP Alo Lye 50 35o TTC CTC GTG GAC AAT CCC TAC ACC GAA AGC TAC TCC TAC TCA PtTe keu Vol ASp Asn Pro Tyr Tier GIu Ser Tyr Set Tyr Ser 70 4OO ACC TGC AAC AGC AAA AAC AAT @CT TGT GAG GCC TTC ATC TGT Thr Cys Asn Ser LYs Ash Asn Alo Cys Glu A]o Phe I]e Cys 90 450 GCC ATT TGC TTC TCA AAG GCC CCA TAC AAC AAG GAG CAC AAG A]o lie Cys Phe Ser Lys Alo Pro Tyr Asn Lys Glu His LyS
300 AAC CTG GAC AGC TGT Aen Leu Asp Ser Cys 60
TGT TCT AAC ACT GAG Cys Ser Aen Thr Glu 80
AAC TGT GAC CGA AAT Ash Cye ASP Arg Aen 100 AAC CTG GAC ACC AAG Asn Leu ASD Thr Lys
II0
120
50(9 AAG TAC TGT TAG AGC TAA GTA TCA CCC Lys Tyr Cys " ' * ### 124
FIG. 1. cDNA and deduced protein sequence of porcine pancreatic preproPLA2. The triangle represents the cleavage site for signal peptidase. Roman numerals denote residues of the activation peptide of proPLA 2 , which is cleaved by trypsin ( a r r o w ) .
can be chosen to solve this problem. First, for expression eukaryotic cells can be used which are capable of in vivo disulfide bond formation. Second, a prokaryote like Escherichia coli can be used as expression host to produce (partly) incorrectly folded protein, and subsequent in vitro refolding and oxidation may then give folded protein with the correct disulfide bonds. Reduction and reoxidation has been shown to be an efficient process both for pancreatic (pro)PLAz and for snake venom PLA 2.19,20 Another problem that is often encountered during heterologous expression deals with the instability of the protein of interest due to cellular proteases.
Expression in Escherichia coli During our experiments aimed at the expression in E. coli, it became evident that porcine pancreatic proPLA2 could not be expressed to any significant levels, either directly in the cytoplasm, or after processing into 19G. J. M. van Scharrenburg, G. H. de Haas, and A. J. Slotboom, Hoppe-Seyler'sZ. Physiol. Chem. 361, 571 (1980). 20 S. Tanaka, Y. Takahashi, N. Mohri, H. Kihara, and M. Ohno, J. Biochem. (Tokyo) 96, 1443 (1984).
218
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[19]
the periplasmic space. For this reason we developed a strategy for the expression of PLA2 as a 59-kDa fusion protein which precipitates inside the cell. The approach combines several published methodsl°: (1) purification of insoluble fusion proteins from the cytoplasm, (2) S-sulfonation and subsequent reoxidation and renaturation of recombinant proteins, and (3) site-specific cleavage of the fusion protein with hydroxylamine or trypsin. Hydroxylamine cleavage of the peptide bond beteen Ash and Gly residues, a sequence which is absent in porcine pancreatic PLA 2, was optimized by the insertion of three consecutive Asn-Gly sequences between proPLA2 and the bacterial leader fragment of the fusion protein. For tryptic cleavage the "natural" cleavage site, namely, the Arg-Ala 1 bond, is used. FQr bovine pancreatic PLA2 an expression system has been published which makes use of a preproPLA 2 construct giving rise to the precipitation of the desired protein inside the cell, and a protocol was given for small-scale purification. 6 In our laboratory E. coli K12 strain AB1157 was used originally 1° as the host organism for the pEX-derived expression vector and for vector plasmid pCI857, carrying the temperature-sensitive h repressor. At a later stage this strain was replaced by strain MC 4100, 21 a strongly growing strain with a chromosomal lacZ deletion. More recently the DNA fragment coding for the truncated Cro-lacZ-proPLA2 fusion • protein • 10has been introduced into vector pUEX, 22 which by itself carries the temperature-sensitive h repressor. Although the protein yields are similar, the main advantage is the improved ease of manipulating one plasmid instead of two. To produce fusion protein in shaking bottles, a fresh overnight culture, grown at 30° in the presence of antibiotics, is diluted 5-fold with warm (41 °) medium. Induction is continued for 3 hr, after which the cells are harvested by centrifugation. In a fermentor (New Brunswick Microferm Fermentor Model MF-114, New Brunswick Scientific Co., New Brunswick, N J) the overnight starter culture is used to inoculate a 10-fold volume of medium. Stirring is set at 400 rpm, and aeration is kept at 300 ml/min per liter medium. Growth is continued at 30° until the OD600 is 0.6 (about 2 hr) after which the temperature is shifted in about 20 min to 41 °. The OD6o0increases to about 3.5, and after 3 hr of induction cells are harvested by centrifugation. Cells are broken by sonication after lysozyme treatment, protein aggregates are isolated from cell lysates by a 30-min centrifugation step at 5000 g; and the protein pellet is washed once with 0.1% Triton X-100. Starting from a 10-liter culture, a typical yield is 200-400 mg
n M. J. Casadaban, J. Mol. Biol. 104, 541 (1976). 22 G. M. Bressan and K. K. Stanley, Nucleic Acids Res. 15, 10056 (1987).
[19]
RECOMBINANT PANCREATICPHOSPHOLIPASEA2
~
219
Inclusionbodies Dissolve
Reducti~/._(. "~eduction/rena tur:~i~ GOOH
2H N ~
~ Tryl:)sin
Hydroxylamine/ Renaturation
H2N~ ' ~
PRO-PLA
COOH
*PRO-PLA
H2N. ~ ~ ) COOH PLA
FIG. 2. Schematic representation of the refolding of PLA2 and cleavage to proPLA2 and active PLA2, respectively. The black arrow represents the trypsin cleavage site, ---(3 a free SH group, ------(3---(3---a disulfide bond, and t2 the Asn-Gly linker.
fusion protein, being the major band on sodium dodecyl sulfate (SDS)polyacrylamide gels. The order of subsequent steps is outlined schematically in Fig. 2. The fusion protein is first sulfonated at 2-5 mg/ml, as described by Tannhauser and Sheraga. 23 The S-sulfo protein is precipitated by 5 volumes of a cold 1% (w/v) acetic acid solution. The precipitate is collected by centrifugation, and the pellet is washed several times with water. The pellet originating from a l-liter culture is dissolved in 20 ml of 8 M urea, 100 mM sodium borate (pH 8.5), 20 mM EDTA. After dilution to 80 ml, reduced and oxidized glutathione are added to final concentrations of 2 and I mM, respectively. Renaturation usually reaches a maximal level within 24 hr at room temperature, as measured in an L-dioctanoyllecithin assay) ° The (partly) renatured fusion protein is assayed for its content of active PLA2 by adding a portion to the micellar assay reaction vessel, followed by l0 /zg of trypsin. Within minutes the tryptic activation is complete as judged from the fact that the recorded hydroxide uptake becomes linear with time. 23 T. W. Tannhauser and H. A. Scheraga, Biochemistry 24, 7681 (1985).
220
PHOSPHOLIPASE STRUCTURE--FUNCTION TECHNIQUES
[19]
After dialysis, preparative tryptic digestion of the renatured fusion protein is done in a buffer containing 20 mM Tris (pH 8) and 5 mM CaCI 2. Trypsin (5/zg/mg fusion protein) is added in the beginning and after I hr. Normally, after 2-3 hr of incubation at room temperature the PLA2 activity reaches a maximum. The mixture is acidified to pH 4.5, dialyzed against 5 mM sodium acetate, pH 4.8, and centrifuged to remove insoluble peptides. Occasionally the pellet may contain residual PLA 2 activity. If so, the pellet is redissolved at pH 8, acidified to pH 4.8, and centrifuged. The combined supernatants are applied to a carboxymethyl-cellulose column (20 ml for 100.mg fusion protein) equilibrated at pH 4.8 with 5 mM sodium acetate. The PLA2-containing fractions are further purified by two consecutive columns: a carboxymethyl-cellulose column at pH 6 and a diethylaminoethyl-cellulose column at pH 7.5-8.5, depending on the isoelectric point of the mutant PLA 2. In Fig. 3 the elution patterns for a Y69F mutant PLA2 are depicted. Starting from a 10-liter culture, the yield of pure PLA2 is 40-90 mg, reflecting a nearly quantitative conversion of 200-400 mg of the 59-kDa fusion protein. ProPLA2 is isolated from the fusion protein by cleavage with hydroxylamine. First, the inclusion bodies are solubilized under reducing conditions, at 5 mg/ml in 6 M guanidine, 50 mM Tris (pH 8), 1 mM EDTA and 5 mM 2-mercaptoethanol, for 1-3 hr under N2. The mixture is then centrifuged to remove insoluble particles, and the supernatant is diluted 10-fold with cold 0.1% acetic acid. The precipitated fusion protein is collected by centrifugation, and the pellet is washed 3 times with dilute acid. The pellet is dissolved, at a protein concentration of 5 mg/ml, in 6 M guanidine, 2 M hydroxylamine, adjusted to pH 8.5 with LiOH. After a 3 hr incubation at 45 °, the reaction is stopped by the addition of 0.1 volume acetic acid followed by dialysis against 1% acetic acid. Insoluble material is removed by centrifugation, and the soluble fraction is adjusted to pH 8 with NaOH. Additions are made to the following final concentrations: guanidine (1.5 M), Tris-HC1 (50 mM, pH 8.0), Ca 2÷ (I0 mM), reduced glutathione (2 mM), and oxidized glutathione (1 mM). Following renaturation and dialysis, proPLA 2 is purified as described above for the active enzyme. From a 10-liter culture about 15 mg of pure proPLA 2 is obtained.
Expression in Yeast
Whereas expression of PLA2 in E. coli does not lead to the production of active (pro)PLA2 because no or incorrect formation of disulfide bonds occurs in the cytoplasm, the yeast Saccharornyces cerevisiae is capable of both in vivo disulfide bridge formation and efficient secretion of heterologous proteins. Therefore, this yeast has been used as a host system for
[19]
RECOMBINANT PANCREATIC PHOSPHOLIPASE A 2
/ z
221
p.4.8 CM
0 10 20 30 40 50 60 70
ilC I1: I-
CM pH 6.0
n-
O
co
d 6
0
A
10
20
30
I I DEAE pH 8.0
0 10 20 30 FRACTION NUMBER
FIG. 3. Elution profiles of Y69F mutant porcine pancreatic PLA 2 after tryptic digestion of fusion protein. The recorded profiles were obtained starting from a 10-liter culture. Phospholipase A 2 activity is indicated by hatched areas. CM, pH 4.8, and CM, pH 6.0: carboxymethyl-cellulose columns run at pH 4.8 and 6.0, respectively; DEAE, pH 8.0: diethylaminoethyl-cellulose column run at pH 8.0. Proteins were eluted from the columns with linear gradients of NaCl of 0-0.5, 0-0.4, and 0-0.2 M, respectively. For details, see text.
the expression and secretion of several mammalian enzymes. Both bovine 7 and porcine 24-26 (pro)PLA2 have been expressed and purified from this organism. In our laboratory constitutive expression of proPLA 2 in S. cerevisiae was obtained after fusing the proPLA2 to the prepro sequence of the yeast a-mating factor. 24On secretion, the fusion protein was cleaved by the KEX2 protease, yielding a 140-amino acid long precursor form of the PLA2. This protein, present in concentrations of about 600/zg/liter 24 C. J. van den Bergh, A. C. A. P. A. Bekkers, P. de Geus, H. M. Verheij, and G. H. de Haas, Fur. J. Biochem. 140, 241 (1987). 25 C. J. van den Bergh, Ph.D. Thesis, University of Utrecht (1989). 26 A. C. A. P. A. Bekkers, P. A. Franken, C. J. van den Bergh, J. Verbakel, H. M. Verheij, and G. H. de Haas, submitted for publication.
222
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[19]
culture supernatant, was easily purified by ion-exchange chromatography. In later experiments 25 we have replaced the strong constitutive promoter of the yeast a-mating factor by the galactose-inducible G A L 7 promoter. The construct furthermore contains the invertase signal sequence preceding the a-mating factor prosequence-proPLA 2 fusion. 26 Cells harboring this construct target proPLA2 into the culture medium to a level between 2.5 and 6 mg/liter. A similar level of production has been observed by Tanaka et a l . 7 for the expression/secretion of bovine pancreatic proPLA 2 by yeast under control of the P H 0 5 promoter. Whereas in our laboratory only proPLA2 was excreted, Tanaka et al. obtained a mixture of precursor and active PLA2. It is not clear whether this partial activation can be explained by the use of different expression constructs or whether it is strain dependent. Purification of yeast proPLA2 from fermentation supernatants of 10 liters or more can be accomplished with the use of ion-exchange chromatography, 24 thereby circumventing the use of Sephadex columns at an early stage of the purification method as was done by Tanaka et al. 7 After removal of the cells by centrifugation, the culture medium is acidified to pH 3.5, and 15 ml of preswollen SP-Sephadex C-25 is added per liter of medium. After gentle stirring for several hours at 4 °, the beads are collected and washed first with 10 mM formate buffer, pH 3.5, and then with 10 mM acetate buffer, pH 5.0. In this step proPLA2 binds nearly quantitatively to the ion-exchange resin, whereas most colored material remains unbound. I
I
I
I
I
I
I
-
.
0.4
:[
0.2~
.z
0 10
20 30 40 FRACTION NUMBER
50
FIG. 4. Purification of yeast proPLA2 by SP-Sephadex chromatography. The supernatant from 10 liters of culture broth of the strain harboring the GAL7 construct encoding native porcine pancreatic PLA2 (see text) was acidified to pH 3.5 and bound to 150 ml SP-Sephadex. After several wash steps the beads were loaded on top of a column containing 250 ml fresh SP-Sephadex. For details, see text.
[20]
ANTIBODIES TO PHOSPHOLIPASES
A2
223
The SP-Sephadex beads are then packed into a column on top of a fresh layer of SP-Sephadex C-25. The column is eluted with a salt gradient from 0 to 0.5 M NaCI. The proPLA2 peak elutes after the majority of the strongly absorbing colored material which forms the large peak seen in Fig. 4. At this stage the proPLA 2 is the major band on SDS-polyacrylamide gel electrophoresis. After dialysis, the proPLA2-containing fractions are further purified on a carboxymethyl-cellulose column at pH 6.0 in 5 mM acetate buffer. The resulting preparation is desalted and freed from traces of colored material on a Sephadex G-50 fine column in 10 mM acetic acid. Yeast proPLA 2 has the same turnover number for monomeric substrates as native proPLA 2 , although its activation peptide in the a-mating factor and in the G A L 7 constructs is 9 amino acids longer than in native proPLA2. Apparently the extra amino acids do not hinder the entrance of substrate to the active site; they are probably fully exposed to the solvent and do not form contacts with any other part of the enzyme. This is also evident from the fact that yeast proPLA2 is as rapidly cleaved by trypsin as is native proPLA 2 . The resulting yeast PLA2 is indistinguishable from authentic pancreatic PLA2, showing that a protein with as many as seven disulfide bridges per 124 amino acid residues is correctly processed by the yeast secretory apparatus. Yeast, rather than E. coli, may therefore be the organism of choice to express PLA 2 mutants with impaired folding properties.
[20] P r e p a r a t i o n o f A n t i b o d i e s to P h o s p h o l i p a s e s A 2 B y MAKOTO M U R A K A M I , KIYOSHI TAKAYAMA, MASATO U M E D A , ICHIRO K U D O ,
and KEIZO INOUE
Introduction P h o s p h o l i p a s e s A 2 play important roles in several biological phenomena such as membrane phospholipid turnover, production of lipid mediators (eicosanoids, platelet-activating factor, etc.),~ and the process of inflammation. 2,3 Phospholipases A 2 a r e classified into two groups in terms z H. van den Bosch, Biochim. Biophys. Acta 6114, 191 (1980). 2 p. Vadas and W. Pruzanski, Lab. Invest. 55, 391 (1986). 3 p. Vadas, S. Wasi, H. Z. Movat, and J. B. Hay, Nature (London) 293, 583 (1981).
METHODS IN ENZYMOLOGY,VOL. 197
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