Expression of an active recombinant lysine 49 phospholipase A(2) myotoxin as a fusion protein in bacteria

ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded co pperhead) that induces necrosis of skeletal muscle. We have previously clon ed and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an e xpression system for production of ACLMT as a fusion protein with maltose b inding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expr ession vector. The cDNA coding for the mature toxin without the signal pept ide was amplified by PCR and subcloned into the pMAL-p2 vector. The new pla smid (pMAL-MT) was used to transform BL21 (DE3) E. coli cells. Culture of t ransformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusio n protein was purified from the bacterial periplasm by affinity chromatogra phy in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very s imilar to that caused by the native myotoxin. (C) 2001 Elsevier Science Ltd . All rights reserved.