A novel method for the purification of porcine phospholipase A2 expressed in E. coli.
Porcine phospholipaseA2 expressed in E. coli as a fusion protein was isolated, renatured and specifically cleaved by trypsin as described in (1). Active phospholipaseA2, was purified to homogeneity on a column of PBE-94 over a pH region 7.4-4.5. Using this method, several phospholipase A2 mutant enzymes have now been purified in a single step and all behaved identically during chromatofocusing. The method will therefore be extremely useful not only for those interested in understanding the structure-function relationships of phospholipaseA2 but also for preparing the enzyme in large quantities for industrial and pharmaceutical purposes.
Bhat
K M
KM
AFRC Institute of Food Research, Reading Laboratory, Berks, U.K.
Sumner
I G
IG
Perry
B N
BN
Collins
M E
ME
Pickersgill
R W
RW
Goodenough
P W
PW
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D008823:Micelles; D008970:Molecular Weight; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014357:Trypsin
D008823:Micelles; D011993:Recombinant Fusion Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin