Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Protein Eng Des Sel. 2011 Aug;24(8):607-16. doi: 10.1093/protein/gzr030. Epub 2011 Jul 5.
Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.
在大肠杆菌中，重组表达蛋白的低蛋白溶解度是阻碍其应用和分析的主要因素。我们使用蛋白质工程技术生成了一种羟腈裂解酶在大肠杆菌中的高度体内可溶性突变体。结构指导的饱和突变导致该低可溶性野生型酶的位置 176、199 和 224 处的单个赖氨酸-脯氨酸突变具有高溶解度。在这些表面保守残基处产生的三赖氨酸-脯氨酸突变体在无细胞提取物中的比活性增加了 8 倍。随机突变还产生了一个组氨酸 103 突变为蛋氨酸的突变体，其活性增加了 18.5 倍。在 37°C 下，上述两种突变都使大肠杆菌中的主要表达形式从不可溶转变为可溶部分。这一发现挑战了在 37°C 下在该宿主中生产重组蛋白的基本原理。以前的野生型低可溶性蛋白通过这些突变现在以可溶性蛋白的形式存在，这也提高了大肠杆菌中总可溶性蛋白的分数。对组氨酸 103 的饱和突变提供了其他具有疏水性取代的高度可溶性突变体。这些突变仅引起较小的二级结构变化，如圆二色性和傅里叶变换红外光谱所确定的，并且对纯化突变体的催化效率略有影响（苯甲醛为 0.82-1.6 倍，扁桃腈为 0.9-1.9 倍）。与野生型相比，突变体在高温和 pH>8 时的稳定性不同。将埋藏的碱性极性残基组氨酸 103 与疏水性氨基酸交换符合酶的整体结构，即在溶剂暴露区域具有亲水性残基，在核心区域具有疏水性残基。
