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

Abstract

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 BL21(DE3). 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.

Identification of lumbrokinase by fibrin-plate method, fibrin-zymography and liquid chromatography mass spectrometry

“…[13] Some lumbrokinases exhibited a highly similar loop and specific pocket (Asn-Asp-iLe-Ala-Leu-Leu), similar to tPA and uPA. [14–16] Besides, the amino acids, asp 507 , ser 532 and gly 534 in tPA, i.e. important substrate recognition sites for tPA and uPA, [17–19]also existed in several lumbrokinases.…”

“…These results were consistent with the report that the protein property of these proteases, by activating the plasminogen into plasmin, both had the activity of degrading the fibrin directly and the activity of degrading the fibrin indirectly. [14] However, the current fibrin plate method used in the potency determination of lumbrokinase only measured its direct fibrinolytic activity with its kinase activity excluding. Accordingly, the current method should be optimized to measure its total activity.…”

Expression of cholera toxin B subunit–lumbrokinase in edible sunflower seeds–the use of transmucosal carrier to enhance its fusion protein's effect on protection of rats and mice against thrombosis

“…The high level of similarity in amino acid sequence among LK proteins indicates that some LK proteins have a comparatively recent common ancestor . The phylogenetic analysis also indicated that most LK genes were closely related to each other . The differences in LK protein sequences between species may be due to the diverse habitats occupied and food resources utilized by the different species of earthworm .…”

“…3 The phylogenetic analysis also indicated that most LK genes were closely related to each other. 4 The differences in LK protein sequences between species may be due to the diverse habitats occupied and food resources utilized by the different species of earthworm. 1 The LK gene used in this study was originally cloned from Eisenia fetida and was codon optimized by Hu et al 5 Currently, LK proteins are widely used as thrombolytic agent in China to treat coronary heart disease, cerebral infarction and deep vein thrombosis.…”

Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus‐Based Single Replicon System Dissolves Fibrin and Blood Clots

“…Various LK genes have been cloned and produced in different expression systems including Escherichia coli (E. coli) [ 7 , 8 ], yeast [ 9 , 10 ], and mammalian [ 11 ] systems. However, results from those investigations have not been promising.…”