Chemical synthesis, molecular cloning, overexpression, and site-directed mutagenesis of the gene coding for pumpkin (Curcubita maxima) trypsin inhibitor CMTI-V.
The gene encoding for a pumpkin (Curcubita maxima) trypsin inhibitor CMTI-V was synthesized chemically. The synthetic gene was prepared from four overlapping oligonucleotides by overlapping extension. The synthetic gene was amplified by polymerase chain reaction, cloned into a T7 expression vector and expressed in Escherichia coli as a fusion protein. The clone, namely 70-1, encoded a fusion protein containing 7 amino acid residues of the N-terminus of the bacterial protein rho 10 and the entire 68 residues of CMTI-V. The wild-type fusion protein constituted approximately 15% of the total bacterial protein mass and was purified to homogeneity in a single step by antibody affinity chromatography. The wild-type fusion protein possesses inhibitory activity toward trypsin and beta-Factor XIIa, but to a lesser extent when compared to the natural CMTI-V. A mutant, T43A, in which threonine at position 43 (P2 position) was replaced by alanine, was constructed. This mutant showed considerably lower specific inhibitory activity toward both trypsin and beta-Factor XIIa.
Wen
L
L
Department of Chemistry, Western Illinois University, Macomb 61455.
Kim
S S
SS
Tinn
T T
TT
Huang
J K
JK
Krishnamoorthi
R
R
Gong
Y X
YX
Lwin
Y N
YN
Kyin
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015956:Factor XIIa / Q000037:antagonists & inhibitors* / Q000187:drug effects; D016368:Frameshift Mutation; D017343:Genes, Plant / Q000235:genetics*; D005813:Genes, Synthetic / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014357:Trypsin / Q000187:drug effects; D014361:Trypsin Inhibitors
C081870:CMTI protein, Cucurbita maxima; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C031809:trypsin inhibitor, Cucurbita sp.; D015956:Factor XIIa; D014357:Trypsin