Cloning and expression of earthworm fibrinolytic enzyme PM(246) in Pichia pastoris.
We have cloned, expressed, and purified a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence revealed a 747bp region containing an intact ORF that encodes a protein of 246 amino acid residues, designated as EFE PM(246). While EFE PM(246) is distinct, its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The recombinant EFE PM(246) was active, showing a fibrinolytic activity of 7.5 x 10(6)U/L in basal salts medium, a higher fibrinolytic activity than those produced in other expression systems. The recombinant EFE PM(246) expressed in basal salts medium was purified by a three-step purification procedure with a recovery rate of about 20%. This is the first report detailing the successful purification of a genetically engineered earthworm fibrinolytic enzyme. The main physiochemical features of the EFE PM(246), including temperature stability, pH resistance, and sensitivity to some protein inhibitors, were also characterized.
Hu
Yan
Y
College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China.
Meng
Xiao-Lin
XL
Xu
Jin-Ping
JP
Lu
Wei
W
Wang
Jian
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004795:Enzyme Stability; D005342:Fibrinolysis / Q000235:genetics*; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D018076:DNA, Complementary; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D012697:Serine Endopeptidases