分子伴侣对毕赤酵母表达单域抗体的影响

Molecular chaperones influence the expression of single-domain antibodies in Pichia pastoris

摘要:

单域抗体(single-domain antibodies, sdAbs)因其低分子量和优异的组织穿透性，在生物医药领域的应用日益广泛。相较于哺乳动物细胞和大肠杆菌表达体系，毕赤酵母表达系统兼具成本低、周期短的特性以及完善的蛋白折叠支持能力。为了提升酵母表达sdAbs的效率，本研究构建了6种分子伴侣(HDPI、YPDI、BZIP、TO、RPPO、Sec)与sdAb的酵母共表达体系。通过圆二色谱解析蛋白二级结构、测定热变性温度(thermal melting temperature, Tm)和蛋白半数变性尿素浓度(concentrations of urea when 50% protein unfolded, CUU)，结合浊度实验和胰蛋白酶消化实验，系统评估了分子伴侣对抗体结构与功能特性的调控作用。结果显示：共表达Sec将sdAb表达水平显著提升了1.68倍；分子伴侣的引入均未改变sdAb的二级结构及抗原结合活性；热稳定性实验中，仅BZIP共表达组观察到热稳定性增强(T_m值提升2.4 ℃)；尿素诱导变性实验中，YPDI共表达组的抗尿素变性能力显著提升(CUU值提高1.2 mol/L)；胰蛋白酶消化实验显示，YPDI组的酶解稳定性最优(3 h后活性保留58.3%)；浊度实验表明，YPDI与BZIP共表达可有效抑制抗体聚集，而HPDI、TO和RPPO则促进了聚集现象。综上所述，本研究揭示了不同分子伴侣对单域抗体理化性质的调控效应具有较显著差异。这些发现为优化单域抗体的生产工艺提供了重要的理论依据和实践参考。

Abstract:

Single-domain antibodies (sdAbs) have garnered increasing attention in the biomedical field due to their low molecular weights and exceptional tissue penetration capacity. Compared with mammalian cell and Escherichia coli expression systems, the Pichia pastoris expression system offers advantages such as low costs, short production cycles, and robust support for protein folding. To enhance the expression efficiency of sdAbs in yeast, we constructed a co-expression system in P. pastoris involving six molecular chaperones (HDPI, YPDI, BZIP, TO, RPPO, and Sec) and the target sdAbs. The effects of these chaperones on the structural and functional properties of sdAb were systematically evaluated through circular dichroism (CD) spectroscopy for secondary structure analysis, measurements of thermal melting temperature (T_m) and concentration of urea when 50% protein unfolded (CUU), turbidity assays, and trypsin digestion experiments. The results demonstrated that: Co-expression of Sec significantly increased the expression level of sdAbs by 1.68 times; None of the chaperones significantly altered the secondary structure or antigen-binding activity of the sdAbs; Only the BZIP co-expression group showed enhanced thermal stability, with a Tm increase of 2.4 ℃; The YPDI group exhibited markedly improved resistance to urea-induced denaturation, with a 1.2 mol/L increase in CUU; In trypsin digestion assays, the YPDI group displayed the highest stability, retaining 58.3% of the enzyme activity after 3 h; Turbidity assays indicated that YPDI and BZIP effectively suppressed antibody aggregation, whereas HPDI, TO, and RPPO promoted aggregation. In conclusion, this study reveals distinct regulatory effects of different molecular chaperones on the physicochemical properties of sdAbs, providing a theoretical foundation and practical insights for optimizing the production processes of sdAbs.