Molecular cloning and characterization of a serine protease-like protein from silkworm (Bombyx Mon)

So Youn Kim, Eon Jeong Jeong, Ki Joon Song, Kwang Sook Park

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

The Bombyx mori (B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQl 18520; protein, AAZ40503) was generated from two separate and overlapping cDNA fragments using sequence homology with Trichoplusia ni azurocidin in a Bombyx EST database (Silkbase! httpV/www.ab.a.u-tokyo.ac.jp/silkbase/). The deduced amino acid sequence of BmSp, which encodes 303 amino acids, shows 44% amino acid identity to A gambiae serine protease (CAA89967), 43% amino acid identity to Sarcophagi peregrina 26-kDa protease, an antibacterial protein and 31% identity to B. mon serine protease-2 (BmSP-2), a potential antiviral protein. Typical features of the BmSp included the serine protease active site triad His / Asp / Ser, three pairs of cysteine residues for disulfide bridges, and three residues, Asp / Gly / Gly, that help to confer trypsin-like specificity to the enzymes. Based on the result of sequence comparison and characterization, our results suggest that the BmSp probably the new subfamily of trypsin-like serine protease. Using RT-PCR and enzyme digestion, the full encoding sequence for BmSp was cloned into the E. coli expression vector pGEX-5X-l. The fusion protein GST-BmSp was effectively expressed in E. coli BL2l(DE3) pLysS as inclusion bodies, and a denaturation and refolding procedure were performed to obtain soluble GST-BmSp. The purified protein was tested for antibacterial activity against Gram-positive and Gram-negative bacteria, but it did not show antibacterial activity in the agar well diffusion assay and liquid growth inhibition assay.

Original languageEnglish
Pages (from-to)387-395
Number of pages9
JournalGenes and Genomics
Volume31
Issue number5
DOIs
Publication statusPublished - 2009 Oct

Keywords

  • Antibacterial activity
  • B.mori
  • Serine protease
  • Serprocidin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

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