Cloning of an alkaline lipase gene from Penicillium cyclopium and its expression in Escherichia coli.
The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles. The cloned gene was 1,480 nucleotides in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides. The open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide, with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases. However, this protein had a low homology with lipases of P. camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level. The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation. A distinct band with a M.W. of 33 kDa was detected on SDS-PAGE. Results of a Western blot analysis and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase. Its catalytic properties were not changed when compared with its natural counterpart.
Wu
Minchen
M
Medical Department, Southern Yangtze University, Wuxi 214063, Jiangsu, PR China.
Qian
Zhikang
Z
Jiang
Peihong
P
Min
Taishan
T
Sun
Chongrong
C
Huang
Weida
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D007438:Introns; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases