Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies.
A synthetic gene encoding the Group II phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA2's were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA2. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA2 demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.
Lathrop
B K
BK
Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville 22908.
Burack
W R
WR
Biltonen
R L
RL
Rule
G S
GS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics*; D054467:Phospholipases A2; D012910:Snake Venoms / Q000201:enzymology*; D012911:Snakes / Q000235:genetics*; D013329:Structure-Activity Relationship
D012910:Snake Venoms; D010741:Phospholipases A; D054467:Phospholipases A2