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A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris

文献类型： 外文期刊

作者： Zheng, Yiheng 1 ; Li, Bingkun 1 ; Zhao, Shida 1 ; Liu, Jiawei 1 ; Li, Ding 1 ;

作者机构： 1.Jiangsu Acad Agr Sci, Inst Vet Immunol & Engn, Nanjing 210014, Peoples R China

2.Henan Univ Sci & Technol, Coll Food & Bioengn, Luoyang 471000, Peoples R China

3.Jiangxi Agr Univ, Coll Biosci & Bioengn, Nanchang 330045, Peoples R China

4.Nanjing Tech Univ, Coll Biotechnol & Pharmaceut Engn, Key Lab Waste Plast Biocatalyt Degradat & Recyclin, Nanjing 211800, Peoples R China

关键词： model nanobodies; antibiotic-free parental plasmids; expression components; gene dosage; molecular chaperones; H55 host

期刊名称：FERMENTATION-BASEL （ 影响因子：3.7； 五年影响因子：4.5 ）

ISSN：

年卷期： 2024 年 10 卷 1 期

页码：

收录情况： SCI

摘要： In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOX alpha and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOX alpha-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future.

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