Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.
A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.
Yue
Chang-Wu
CW
College of Life Science, Sichuan Key Laboratory of Molecular Biology & Biotechnology, Sichuan University, Chengdu, PR China.
Zhang
Yi-Zheng
YZ
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D013696:Temperature; D013712:Tenebrio / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D021301:Antifreeze Proteins; D018076:DNA, Complementary