[Cloning, expression of fibrinolytic enzyme gene Efp-I from Eisenia fetida in Escherichia coli and activity analysis].
Earthworm fibrinolytic enzyme (EFE) is a group of protease having fibrinolytic and plasminogen-activator activities isolated from earthworm. Molecular biology research showed that there were 21 EFE coding sequences, in which only one sequence, AY438624, whose translated protein had similar N-terminal amino-acid sequence to EfP-I purified from Eisenia fetida. To obtain coding sequence of EfP-I , we designed specific primers according to 5' and 3' sequences of AY438624. A new DNA sequence was obtained by RT-PCR, sequence analysis showed that the protein translated from the coding sequence had identical N-terminal amino-acid sequence with EfP-I purified from Eisenia fetida and Lumbricus rubellus. Analysis by using ScanProsite prediction programs proved that the sequence had high similarity to AY438624 and belonged to trypsin family of serine protease. But there was difference between two sequences, that was there was a domain of characteristic amino acids of N-glycosylation site Asn-Xaa-Ser/Thr(N-x-S/T)in the new sequence (DQ418454). Then the expressed vector pMAL-c2X-Efp-I was constructed by cloning the gene into the plasmid pMAL-c2X, and was transformed to E. coli TB1. After induction and expression of the recombinant, the product MBP-EfP-I was purified by MBP affinity chromatography. Western blotting analysis showed that the product reacted with both anti-MBP and anti-EfP-I -1 serum. Casein plate test and fibrin plate test showed that the protein expressed had fibrinolytic activity.
Zhao
Xiao-Yu
XY
College of Life Sciences, Hebei University, Baoding 071002, China. uyoaix@yahoo.com.cn
Li
Xiao-Xia
XX
Hou
Yan
Y
Jing
Tian-Yu
TY
D000818:Animals; D055162:Biocatalysis; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D015971:Gene Expression Regulation, Enzymologic; D009835:Oligochaeta / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D005337:Fibrin; D057057:Serine Proteases