Genomic and cDNA cloning, characterization of Delonix regia trypsin inhibitor (DrTI) gene, and expression of DrTI in Escherichia coli.
Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.
Hung
Chih-Hung
CH
Department of Medical Technology, Yuanpei University, Taiwan, ROC. chihhung@mail.yust.edu.tw
Peng
Pei-Hua
PH
Huang
Chou-Chun
CC
Wang
Hai-Lung
HL
Chen
Yu-Jen
YJ
Chen
Yuan-Liang
YL
Chi
Lang-Ming
LM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D007887:Fabaceae / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D018745:Genome, Plant / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors