In silico cloning of EfP-0, a novel earthworm fibrinolytic enzyme gene, and verification of its coding region by RT-PCR

There are four different types of N-terminal amino sequences (F-I-0, F-I, F-II, F-III) in the multicomponents of earthworm fibrinolytic enzymes (EFE). In GenBank, 21 nucleic acid sequences of EFE have been reported. Among them, most of the N-terminal amino sequences belong to the F-III type, and few belong to the F-II type. Only one is similar to the F-I type, but none to F-I-0. In this research, an attempt was made to obtain the gene encoding component F-I-0 of EFE using the bioinformatics tools. On the basis of the N-terminal amino acid sequence VVGGSDTTIGQYPHQL of the F-I-0 type from Lumbricus rubellus, a nucleic acid sequence was obtained by in silico cloning from dbEST of Lumbricidae using the software DNAMAN. A new gene of EFE from Eisenia foetida was successfully obtained by RT-PCR using specific primers designed according to this sequence. The new gene named EfP-0 was cloned in pMAL-c2x and expressed as the fusion protein MBP-EfP-0 in the supernatant of the lysate. The fusion protein MBP-EfP-0 purified by affinity chromatography exhibited hydrolytic activity on casein plates. Sequencing result shows that EfP-0 has 678 bp and encodes a protein of 225 amino acids. The protein is a serine protease belonging to the trypsin family. It has similar amino acid composition to F-I-0. BLAST in GenBank shows that the similarity is lower than 40% between EfP-0 gene and other EFE genes. From the above-mentioned facts, it is concluded that EfP-0 gene of EFE is a novel gene and is first reported in this study, and its accession number for GenBank is DQ836917.