Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression
Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression
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DOI：
10.1016/j.cbi.2019.04.012
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发表时间：
2019-06-01
影响因子：
5.1
通讯作者：
Zheng, Fang
中科院分区：
文献类型：
--
作者：
Chen, Xiabin;Deng, Jing;Zheng, Fang
人丁基胆碱酯酶（BChE）被认为是一种安全有效的解毒有机磷（OP）神经毒剂的蛋白质。其合理设计的突变体具有显著提高的对可卡因的催化活性，被称为可卡因水解酶（CocHs），被认为是治疗可卡因滥用最有希望的候选药物。然而，如何高效地生产具有较长生物半衰期的活性重组BChE和CocHs是一个巨大的挑战。在本研究中，我们从一种有前景的CocH，即CocH3（即人BChE的A199S/F227A/S287G/A328W/Y332G突变体）开始，设计了一种n端融合蛋白Fc (M3)-(PAPAP)(2)-CocH3，该蛋白将人IgG1的Fc融合到CocH3的n端，并通过在两个蛋白结构域之间插入连接体进行进一步优化。在不降低酶活性的情况下，在中国仓鼠卵巢（CHO）细胞中表达的Fc(M3)-(PAPAP)(2)- coch3不仅在大鼠体内具有105 +/- 7 h的生物半衰期，而且蛋白表达量高。特别是，在相同的实验条件下，Fc(M3)-(PAPAP)(2)-CocH3在CHO细胞中的蛋白表达量比CocH3提高了21倍。鉴于Fc(M3)-(PAPAP)(2)- coch3不仅对可卡因具有高的催化活性和较长的生物半衰期，而且蛋白表达量高，本研究报道的这种新的蛋白实体将是治疗可卡因过量和成瘾的更有希望的候选蛋白。
Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a similar to 2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc (M3)-(PAPAP)(2)-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)(2)-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 +/- 7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)(2)-CocH3 has a similar to 21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)(2)-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.