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

Title
Molecular cloning and characterization of a serine protease-like protein from silkworm (Bombyx Mon)
Authors
Kim S.Y.Jeong E.-J.Song K.-J.Park K.S.
Ewha Authors
김소연
Issue Date
2009
Journal Title
Genes and Genomics
ISSN
1976-9571JCR Link
Citation
vol. 31, no. 5, pp. 387 - 395
Indexed
SCIE; SCOPUS; KCI WOS 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.
DOI
10.1007/BF03191257
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연구기관 > 세포신호전달계바이오의약연구센터 > Journal papers
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