High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding.
Microbial lipases are widely used for biotechnological applications. In this study we have cloned and sequenced the lipase and lipase specific foldase genes of a Pseudomonas sp., which was isolated from Southern Iran. The lipase was composed of 936 bp which encoded 311 amino acids and the lipase specific foldase gene consisted of 1008 bp which encoded 336 amino acids. The low amount of recombinant lipase was expressed as an active enzyme in Escherichia coli harboring a plasmid with the clustered lipase and lipase specific foldase genes. To increase the enzyme expression level, the lipase and lipase specific foldase genes subcloned into two separate expression vectors. The lipase was expressed as inactive inclusion bodies under the control of the strong T7 promoter. Inclusion bodies were dissolved in 8M urea and 1mM dithiothreitol (DTT) and purified using Ni-nitrilotriacetic acid column. Subsequently, purified lipase diluted in 20mM phosphate buffer (pH 7) containing the lipase specific foldase which was expressed in another clone of E. coli. In the presence of foldase, it was possible to achieve active lipase with a specific activity of up to 240 IU/mg using a simple refolding procedure. Moreover, the effect of different concentrations of various additives was investigated on the refolding of denatured lipase. The best yield of 70 IU/ml with the specific activity of 3000 IU/mg were obtained after incubation of denatured enzyme in a refolding buffer containing lipase specific foldase (0.005 mg/ml), 1M NaCl and 10% glycerol at 4 degrees C.
Akbari
Neda
N
Department of Microbiology, Faculty of Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Khajeh
Khosro
K
Rezaie
Sasan
S
Mirdamadi
Saeed
S
Shavandi
Mahmoud
M
Ghaemi
Nasser
N
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000201:enzymology; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D011549:Pseudomonas / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase