Overexpression of a Rhizopus delemar lipase gene in Escherichia coli.
A cloned complementary deoxyribonucleic acid encoding the precursor polypeptide of an extracellular lipase from the fungus Rhizopus delemar was altered by site-directed mutagenesis to generate deoxyribonucleic acid fragments that specifically code for the polypeptides of the proenzyme and the mature form of the lipase. Attempts to produce these polypeptides in enzymatically active form in Escherichia coli revealed toxic effects toward the host. Therefore the polypeptides were expressed as inactive and insoluble forms in the cytoplasm of E. coli BL21 (DE3) cells using plasmid vector pET11-d. With this tightly regulated high-level expression system, lipase and prolipase polypeptides were produced to estimated levels of up to 21% and 15%, respectively, of total cellular protein. The insoluble polypeptides were solubilized in 8 M urea. Refolding into active forms was achieved by treatment with the redox system cystine/cysteine and dilution. Refolded mature lipase was purified to homogeneity by affinity and ion exchange chromatography. The enzyme had a specific activity comparable to that of lipase from the fungal culture. The quantities of pure enzyme obtained from a 1-L culture of E. coli exceeded those obtained from the fungal culture by a factor of at least 100. Refolded recombinant prolipase was purified essentially to homogeneity and had a specific activity similar to that of the mature enzyme. Its pH optimum was 7.5, rather than the pH 8 determined for recombinant mature lipase and for the enzyme purified from the fungal culture. Recombinant prolipase retained activity after 15 min incubation at 65 degrees C, while mature lipase retained activity only up to 45 degrees C.
Joerger
R D
RD
Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia, Pennsylvania 19118.
Haas
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D003553:Cystine; D004247:DNA / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics*; D012995:Solubility; D014508:Urea
D011994:Recombinant Proteins; D003553:Cystine; D014508:Urea; D004247:DNA; D008049:Lipase; D003545:Cysteine; D004229:Dithiothreitol