Expression, purification and characterization of a three-domain Kazal-type inhibitor from silkworm pupae ( Bombyx mori)

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 146; no. 2; pp. 234 - 240
• Main Authors: Zheng, Qing-Liang, Chen, Jian, Nie, Zuo-Ming, Lv, Zheng-Bing, Wang, Dan, Zhang, Yao-Zhou
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.02.2007
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Bombyx - genetics; Bombyx - metabolism; Bombyx mori; Chymotrypsin - antagonists & inhibitors; Chymotrypsin - metabolism; Cloning, Molecular; DNA, Complementary - chemistry; DNA, Complementary - genetics; Dose-Response Relationship, Drug; Escherichia coli; Expression; Kazal-type; Molecular Sequence Data; Molecular Weight; Protein Structure, Tertiary; Pupa - genetics; Pupa - metabolism; Purification; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Sequence Alignment; Sequence Analysis, DNA; Serine protease inhibitor; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - genetics; Serine Proteinase Inhibitors - pharmacology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subtilisin inhibitor; Subtilisins - antagonists & inhibitors; Subtilisins - metabolism; Thrombin - metabolism; Kazal-type; Bombyx mori; Purification; Expression; Serine protease inhibitor; Subtilisin inhibitor
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2006.10.106

Serine protease inhibitors are essential for host physiological and immunological activities in insects. Analyzing the amino-acid sequence of a cDNA coding for a serine protease inhibitor in Bombyx mori (BmSPI), we found that BmSPI contained three homologous domains with a conserved sequence of C–X 3–C–X 9–C–X 6–Y–X 7–C–X 3–C–X 11–C similar to that of Kazal-type serine protease inhibitors, suggesting BmSPI as a new member of the Kazal-type serine protease inhibitor family. To characterize the three-domain Kazal-type inhibitor from silkworm pupae, the recombinant protein was expressed in Escherichia coli BL21 (DE3) Star. After purification with affinity and reversed-phase chromatographies, the recombinant BmSPI with a molecular mass of 33.642 Da was shown to be a specific subtilisin A inhibitor. Further studies indicated that the K i value of the recombinant BmSPI was 3.35 nM and the inhibitor seemed to form a 1:1 complex with subtilisin A. This is a first description of the structure and characterization of Kazal-type inhibitor with three domains cloned from silkworm pupae, B. mori.