Sarkar Mohosin, Harsch Christina Keventzidis, Matic George T, Hoffman Kathryn, Norris Joseph R, Otto Tamara C, Lenz David E, Cerasoli Douglas M, Magliery Thomas J
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
J Lipids. 2012;2012:610937. doi: 10.1155/2012/610937. Epub 2012 Jun 7.
Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.
对氧磷酶-1（PON1）是一种血清蛋白，其活性与心血管疾病易感性以及有机磷（OP）化合物中毒有关。它也可能参与固有免疫，并且是开发OP农药和神经毒剂催化性生物清除剂的一个潜在先导分子。在大肠杆菌中表达的人PON1大多存在于不溶性部分，这促使人们构建可溶性变体，如G2E6，它与huPON1相比有50多个突变。我们研究了三组旨在以较少的总体变化使huPON1溶解的突变对其溶解性、活性和稳定性的影响：N端前导序列的缺失、假定的高密度脂蛋白（HDL）结合位点的极性突变，以及从huPON1到G2E6过程中变得更具极性的残基子集的选择。所有三组突变都增加了huPON1的溶解性；HDL结合突变体对溶解性影响最大，但也最大程度地降低了活性和稳定性。基于G2E6的极性突变，我们对一种对环沙林（GF）具有高活性的PON1工程变体进行了“人源化”，发现它对GF仍然非常活跃，并且与人类序列有更高的相似性。
