Cloning, Sequencing and Expression of Novel Serine Protease from Rockworm Marphysa sanguinea

Abstract

To identify and characterize a serine protease (SP) enzyme related gene in Rockworm (Marphysa sanguinea) this study was conducted. Total RNA was isolated from the digestive tract of M. sanguinea, and partial fragments were amplified by degenerate polymerase chain reaction (PCR). Gene-specific primers were designed after partial fragment sequencing, and full-length cDNA was amplified by Rapid Amplification of cDNA Ends (RACE). Full-length cDNA was sequenced and entire sequence possessed 925 bp (SP1), 974 bp (SP2), and the open reading frame (ORF) was 798 bp (SP1) and 780 bp (SP2). SP1, SP2 have 265, 259 amino acid and are composed of the signal-, pre-, and mature peptide. It also has a catalytic triad of histidine, aspartic acid, and serine, and contains the conserved sequence GDSGGP. These results suggest that SP1 and SP2 belong to the serine protease family. Only the mature form was amplified on the basis of the ORF, and cloned into the pET-22b (+) vector and transformed into Escherichia coli BL21 (DE3) for expression and purification. The size of the expressed protein was about 27 kDa and 26 kDa, and enzyme purification was carried out using Ni-IDA column. To measure the fibrinolytic activity of that purified enzyme fibrin plate method was performed, and the formation of a clear zone confirmed fibrinolytic activity. Therefore, these results of a specific gene identification and characterization will be helpful in future research in Rockworm.