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 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.
Giuliani
C D
CD
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, 13565 São Carlos, SP, Brazil.
Iemma
M R
MR
Bondioli
A C
AC
Souza
D H
DH
Ferreira
L L
LL
Amaral
A C
AC
Salvini
T F
TF
Selistre-de-Araujo
H S
HS
D018528:ATP-Binding Cassette Transporters; D017836:Agkistrodon / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015723:Gene Library; D005822:Genetic Vectors; D007273:Injections, Intramuscular; D007544:Isopropyl Thiogalactoside / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008320:Maltose / Q000302:isolation & purification; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D009004:Monosaccharide Transport Proteins; D018482:Muscle, Skeletal / Q000187:drug effects / Q000473:pathology; D009336:Necrosis; D009498:Neurotoxins / Q000378:metabolism* / Q000633:toxicity; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014118:Toxins, Biological / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D014170:Transformation, Genetic
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003435:Crotalid Venoms; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014118:Toxins, Biological; C461599:maltose transport system, E coli; D007544:Isopropyl Thiogalactoside; D008320:Maltose; D010741:Phospholipases A; D008239:Lysine