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 journalArticle

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 Jan 1

Fingerprint

Bombyx
Cloning
Molecular Cloning
Serine Proteases
Proteins
Amino Acids
Escherichia coli
Assays
Morus
Tokyo
Inclusion Bodies
Expressed Sequence Tags
Nucleic Acid Databases
Enzymes
Viperidae
Sequence Homology
Denaturation
Gram-Negative Bacteria
Disulfides
Trypsin

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Molecular cloning and characterization of a serine protease-like protein from silkworm (Bombyx Mon). / Kim, So Youn; Jeong, Eon Jeong; Song, Ki-Joon; Park, Kwang Sook.

In: Genes and Genomics, Vol. 31, No. 5, 01.01.2009, p. 387-395.

Research output: Contribution to journalArticle

Kim, So Youn ; Jeong, Eon Jeong ; Song, Ki-Joon ; Park, Kwang Sook. / Molecular cloning and characterization of a serine protease-like protein from silkworm (Bombyx Mon). In: Genes and Genomics. 2009 ; Vol. 31, No. 5. pp. 387-395.
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