Sample Preparation and Physicochemical Characterization of Rat Lipocalin 11 Nonconservative Cysteins Double Mutant
- 化学化工－已发表论文 
lIPOCAlIn 11蛋白是新发现的一个lIPOCAlIn分泌型蛋白家族成员,与男性生殖密切相关。大鼠lIPOCAlIn 11(rlCn11)蛋白中4个半胱氨酸往往不能正确配对,为重组蛋白样品的制备带来困难。该研究通过序列比对和同源模建的方法获得rlCn11蛋白非保守半胱氨酸的位点,构建了rlCn11蛋白野生型与C59A/C156A突变体的表达系统和纯化方法。通过分子筛和圆二色谱分析rlCn11蛋白的聚集形态和结构特征,并用荧光滴定的方法研究了rlCn11蛋白与荧光探针AnS的结合能力。结果表明:(1)rlCn11蛋白非保守半胱氨酸残基的突变有利于减少蛋白的错误折叠和聚集,提高重组蛋白的产量;(2)rlCn11 C59A/C156A突变体的空间结构良好并能结合疏水性配体;(3)rlCn11蛋白CyS59、CyS156双点突变不显著地改变rlCn11蛋白的空间结构及其与配体的结合能力。该工作为rlCn11蛋白的空间结构解析和生理功能的研究奠定了基础。Lipocalin 11 is a novel secretory protein of the lipocalin family and is potentially related to male reproduction.Misfolding due to the four wrongly paired cysteins in rat lipocalin 11(rLcn11) makes it difficult to prepare the recombinant protein sample.The present work in our lab is to explore two nonconservative cysteins in rLcn11 using sequence alignment and homology modeling.The plasmids of the wild type rLcn11 and its C59A/C156A mutant were constructed and then expressed and purified rLcn11 proteins in Escherichia coli.The oligomeric status and structural characteristics of the protein were analyzed by size-exclusion chromatography and circular dichroism spectroscopy.Ligand-binding abilities were assessed using fluorescence titration experiments by the fluorescent probe ANS.Our results indicate that:(1) The mutation of two nonconservative cysteins could ameliorate the misfolding and aggregation problem with rLcn11,and thus improve the yield of the recombinant protein yield.(2) The rLcn11 C59A/C156A mutant possesses a well-folded structure and a moderate hydrophobic ligand-binding affinity.(3) Mutation of either Cys59 or Cys156 does not significantly change the structural characteristics and ligand-binding affinity of rLcn11.Our work will be of benefit to three-dimensional structure determination and function assay for rLcn11.