In silico cloning of Efp-0, a novel earthworm fibrinolytic enzyme gene and verification of its coding region by RT-PCR.
- Author:
Xiao-Yu ZHAO
1
;
Shan GAO
;
Da-Ling CUI
;
Feng-Ting GENG
Author Information
1. College of Life Sciences, Hebei University, Baoding 071002, China. uyoaix@yahoo.com.cn
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Animals;
Cloning, Molecular;
Computational Biology;
Databases, Genetic;
Endopeptidases;
biosynthesis;
chemistry;
genetics;
metabolism;
Escherichia coli;
genetics;
Expressed Sequence Tags;
metabolism;
Molecular Sequence Data;
Oligochaeta;
enzymology;
genetics;
Open Reading Frames;
genetics;
Reverse Transcriptase Polymerase Chain Reaction
- From:
Chinese Journal of Biotechnology
2006;22(6):897-901
- CountryChina
- Language:Chinese
-
Abstract:
There are four different types of N-terminal amino acid 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 acid sequences belong to the F-III type,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 we hoped to obtain the gene encoding component F-I-0 of EFE by the bioinformatics tools. Based on 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 lysate. The fusion protein MBP-EfP-0 purified by affinity chromatography had hydrolytic activity on casein plate. Sequencing result shows, EfP-0 has 678bp and encodes a protein of 225 amino acids. The protein is a serine protease belonging to trypsin family. It has similar amino acid composition to F-I-0. BLAST in GenBank shows that the similarity is lower than 40% between EJP-0 gene and other EFE genes. By this we conclude that EfP-0 gene of EFE is a novel gene and it is the first time to be reported, its accession number for Genbank is DQ836917.
