Yue Chang-Wu, Zhang Yi-Zheng
College of Life Science, Sichuan Key Laboratory of Molecular Biology & Biotechnology, Sichuan University, Chengdu, PR China.
Mol Biol Rep. 2009 Mar;36(3):529-36. doi: 10.1007/s11033-008-9210-y. Epub 2008 Feb 7.
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.
通过逆转录聚合酶链反应（RT-PCR）从黄粉虫幼虫中克隆出一种新型抗冻蛋白cDNA。339 bp的编码片段编码一个由112个氨基酸残基组成的蛋白质，并与表达载体pET32a和pTWIN1融合。将得到的表达质粒分别转化到大肠杆菌菌株BL21(DE3)、ER2566和Origami B(DE3)中。采用了几种策略在不同的表达系统中表达具有抗冻活性的高度二硫键结合的含β-螺旋蛋白。获得了一种在细菌中生产重折叠且具有活性的黄粉虫抗冻蛋白的方案。
