Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP-Binding Cassette Transporters*
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Agkistrodon / genetics*
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Agkistrodon / metabolism
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Animals
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics*
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Bacterial Proteins / isolation & purification
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Carrier Proteins / chemistry
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Chromatography, Affinity
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Chromatography, Gel
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Crotalid Venoms / enzymology*
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Crotalid Venoms / genetics
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Escherichia coli Proteins*
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Gene Library
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Genetic Vectors*
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Injections, Intramuscular
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Isopropyl Thiogalactoside / metabolism*
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Lysine / chemistry
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Maltose / isolation & purification
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Maltose-Binding Proteins
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Membrane Proteins / biosynthesis*
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Membrane Proteins / chemistry
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Membrane Proteins / genetics*
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Membrane Proteins / isolation & purification
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Monosaccharide Transport Proteins*
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Muscle, Skeletal / drug effects
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Muscle, Skeletal / pathology
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Necrosis
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Neurotoxins / metabolism*
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Neurotoxins / toxicity
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Phospholipases A / genetics*
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Phospholipases A / isolation & purification
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Phospholipases A / toxicity*
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Plasmids / genetics*
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Polymerase Chain Reaction
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Recombinant Fusion Proteins* / genetics*
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Recombinant Fusion Proteins* / isolation & purification
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics*
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Toxins, Biological / isolation & purification*
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Toxins, Biological / metabolism
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Toxins, Biological / toxicity
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Transformation, Genetic
Substances
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ATP-Binding Cassette Transporters
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Bacterial Proteins
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Carrier Proteins
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Crotalid Venoms
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Escherichia coli Proteins
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Maltose-Binding Proteins
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Membrane Proteins
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Monosaccharide Transport Proteins
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Neurotoxins
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Recombinant Fusion Proteins
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Recombinant Proteins
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Toxins, Biological
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maltose transport system, E coli
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Isopropyl Thiogalactoside
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Maltose
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Phospholipases A
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Lysine