High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

毕赤酵母 分泌物 重组DNA 高尔基体 分泌途径 细胞生物学 毕赤酵母 分泌蛋白 化学 生物化学 生物 内质网 基因
作者
Jia Li,Qinan Shao,Yulong Xiang,Jianghua Li,Jian Chen,Guocheng Du,Zhen Kang,Yang Wang
出处
期刊:Biotechnology Journal [Wiley]
卷期号:19 (5)
标识
DOI:10.1002/biot.202400098
摘要

Abstract Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis‐sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty‐two single‐mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer‐aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1‐P6 showed a remarkable 114% increase in the catalytic rate constant ( k cat ), a 137% decrease in the Michaelis constant ( K m ), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1‐P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1‐P6 was produced by the secretion‐enhanced P. pastoris chassis to 199.6 ± 20 mg L −1 with a specific activity of 96 ± 0.32 U mg −1 , resulting in a total enzyme activity of 19137 ± 1131 U L −1 , demonstrating significant potential for industrial applications.
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