Molecular Cloning, Sequencing, and Expression of a Fibrinolytic Serine-protease Gene from the Earthworm Lumbricus rubellus

Abstract

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro-and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat's venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro-and mature regions.

Functional expression of an earthworm fibrinolytic enzyme in Escherichia coli

“…The result confirmed our supposition that the E. coli expression system could not recognize the endogenous signal peptide of F-III-2, which resulted in no activation peptides. The EFE F6 of L. rubellus with or without signal peptide, produced in the E. coli system, had similar fibrinolytic activities in vitro or in vivo assay (Cho et al 2004). The reason for these results is that the expressed F6 with signal peptide was converted in the intestine to active forms when it was assayed (Cho et al 2004).…”

“…The EFE F6 of L. rubellus with or without signal peptide, produced in the E. coli system, had similar fibrinolytic activities in vitro or in vivo assay (Cho et al 2004). The reason for these results is that the expressed F6 with signal peptide was converted in the intestine to active forms when it was assayed (Cho et al 2004). Compared with the signal peptide, poly His tags at the N-terminus of the expressed protein showed no significant effect on the fibrinolytic activity (Fig.…”

“…The genes encoding F-III-2, PM 246 of L. rubellus and F238 of E. fetida have been cloned and expressed with fibrinolytic activity in Pichia pastoris (Sugimoto and Nakajima 2001;Hu et al 2005;Zhao et al 2006). In addition, certain EFEs such as PV242 of L. bimastus, fraction 6 (F6) of L. rubellus and EFE-3 of E. fetida have been produced in E. coli systems (Xu et al 2002;Cho et al 2004;Dong et al 2004).…”

Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus

“…The genes encoding strong fibrinolytic enzymes from these earthworms have been identified (Dong et al 2004). High-throughput production of these enzymes by recombinant DNA technology has been conducted in Escherichia coli (Cho et al 2004 and Xu et al 2010) and Pichia pastoris (Ge et al 2005 and Sugimoto et al 2005). The recombinant enzymes expressed strong fibrinolytic activity both in vitro (Sugimoto et al 2005) and in vivo in rats via oral administration (Cho et al 2004).…”

“…High-throughput production of these enzymes by recombinant DNA technology has been conducted in Escherichia coli (Cho et al 2004 and Xu et al 2010) and Pichia pastoris (Ge et al 2005 and Sugimoto et al 2005). The recombinant enzymes expressed strong fibrinolytic activity both in vitro (Sugimoto et al 2005) and in vivo in rats via oral administration (Cho et al 2004). Crystallographic data of two components of L. rubellus lumbrokinase were obtained, revealing the structure determinants of their catalytic mechanisms (Tang et al 2002 and Wang et al 2005).…”