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KMID : 0381120090310050387
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Genes and Genomics
2009 Volume.31 No. 5 p.387 ~ p.395
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Molecular Cloning and Characterization of a Serine Protease-Like Protein from Silkworm (Bombyx Mori)
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Kim So-Youn
Jeong Eon-Jeong
Song Ki-Joon
Park Kwang-Sook
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Abstract
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The Bombyx mori (B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQ118520; protein, AAZ40503) was generated from two separate and overlapping cDNA fragments using sequence homology with Trichoplusia ni azurocidin in a Bombyx EST database (Silkbase; http://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. mori 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-1. The fusion protein GST-BmSp was effectively expressed in E. coli BL21(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.
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KEYWORD
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serine protease, B. mori, serprocidin, antibacterial activity
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