Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor.
The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.
Chen
X M
XM
Shanghai Institute of Biochemistry, Academia Sinica, People's Republic of China.
Qian
Y W
YW
Chi
C W
CW
Gan
K D
KD
Zhang
M F
MF
Chen
C Q
CQ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D017343:Genes, Plant; D005813:Genes, Synthetic; D005818:Genetic Engineering; D005822:Genetic Vectors; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000138:chemical synthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D014361:Trypsin Inhibitors / Q000138:chemical synthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C068347:Trichosanthes trypsin inhibitor; D014361:Trypsin Inhibitors; D003488:Cyanogen Bromide