Expression, purification and characterization of a three-domain Kazal-type inhibitor from silkworm pupae (Bombyx mori).
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.
Zheng
Qing-Liang
QL
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, China.
Chen
Jian
J
Nie
Zuo-Ming
ZM
Lv
Zheng-Bing
ZB
Wang
Dan
D
Zhang
Yao-Zhou
YZ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D011679:Pupa / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D013381:Subtilisins; D002918:Chymotrypsin; D013917:Thrombin