Cloning, overexpression, refolding, and purification of the nonspecific phospholipase C from Bacillus cereus.
Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. B. cereus PLC has been overexpressed with its signal sequence in Escherichia coli using a T7 expression system. The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 M urea. Renaturation was initiated by gradual removal of urea and addition of zinc ions. The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 M. Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 M. The folded protein was purified by Q-Sepharose Fast flow chromatography to yield a preparation > 99% pure. The final yield of active enzyme was 30-40 mg per liter of culture. The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC from B. cereus. Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure.
Tan
C A
CA
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Hehir
M J
MJ
Roberts
M F
MF
D001409:Bacillus cereus / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014508:Urea
D010743:Phospholipids; D011994:Recombinant Proteins; D014508:Urea; D010738:Type C Phospholipases