Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving ProteinReport as inadecuate




Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein - Download this document for free, or read online. Document in PDF available to download.

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm Eisenia fetida by RT-PCR amplification. The gene designated as CST1 GenBank No. AY840996 was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type tPA, urokinase uPA, and vampire bat DSPAα1 plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21DE3. The molecular mass of recombinant CST1 rCST was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage 65.7% of blood clot lysis was observed when blood clot was treated with 80 mg-mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units-mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.



Author: GangQiang Li , Kevin Yueju Wang , DaHui Li, Nan Wang, DeHu Liu

Source: http://plos.srce.hr/



DOWNLOAD PDF




Related documents