isopropyl-thiogalactoside and Necrosis

ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned 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 expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography 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 similar to that caused by the native myotoxin.

Topics: Agkistrodon; Animals; ATP-Binding Cassette Transporters; Bacterial Proteins; Carrier Proteins; Chromatography, Affinity; Chromatography, Gel; Crotalid Venoms; Escherichia coli; Escherichia coli Proteins; Gene Library; Genetic Vectors; Injections, Intramuscular; Isopropyl Thiogalactoside; Lysine; Maltose; Maltose-Binding Proteins; Membrane Proteins; Monosaccharide Transport Proteins; Muscle, Skeletal; Necrosis; Neurotoxins; Phospholipases A; Plasmids; Polymerase Chain Reaction; Recombinant Fusion Proteins; Recombinant Proteins; Toxins, Biological; Transformation, Genetic