Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli.
The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA.
de Geus
P
P
van den Bergh
C J
CJ
Kuipers
O
O
Verheij
H M
HM
Hoekstra
W P
WP
de Haas
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D010740:Phospholipases / Q000096:biosynthesis*; D010741:Phospholipases A / Q000096:biosynthesis* / Q000235:genetics; D054467:Phospholipases A2; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000494:pharmacology
D006898:Hydroxylamines; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019811:Hydroxylamine; D004247:DNA; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin