High-throughput microdroplet screening reveals chaperone- and time-dependent enhancement of recombinant papain folding and activity in Escherichia coli

IF 3.6 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal Pub Date : 2026-03-01 Epub Date: 2025-12-06 DOI:10.1016/j.bej.2025.110042

Shimiao Chen , Yan Chen , Bin Shan , Yanyan Li , Fuhai Zheng , Yican Luo , Qinyu Lu

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Abstract

Recombinant papain production in Escherichia coli is limited by misfolding, aggregation, and host toxicity. Using a high-throughput microdroplet microbial culture (MMC) platform, we monitored chaperone-assisted folding and identified a transient “lysis window” during mid-log phase when activity peaked at 818.2 U/mg, doubling conventional yields. Co-expression with the Trigger Factor (TF) chaperone was most effective, whereas combining co-translational TF with the post-translational GroEL/ES system caused kinetic incompatibility, leading to massive aggregation and reduced activity (365.6 U/mg). Structural analyses revealed that optimal activity correlates with conformational flexibility rather than a static structure, a feature imparted by TF. Optimizing temporal dynamics and chaperone coordination is therefore essential for producing complex proteins. MMC provides a powerful platform for dissecting these folding pathways; in the redox-engineered E. coli SHuffle strain, this correct folding was synergistically enhanced, dramatically increasing soluble yields (to 450.2 mg/L) while maintaining high specific activity (857 U/mg) and confirming that an oxidizing cytoplasm is key for efficient production.

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高通量微滴筛选揭示了大肠杆菌中重组木瓜蛋白酶折叠和活性的伴侣性和时间依赖性增强

重组木瓜蛋白酶在大肠杆菌中的生产受到错误折叠、聚集和宿主毒性的限制。利用高通量微滴微生物培养（MMC）平台，我们监测了伴侣辅助折叠，并在中期发现了一个短暂的“裂解窗口”，当活性达到818.2 U/mg时，产量是常规产量的两倍。与触发因子（Trigger Factor， TF）伴侣共表达最有效，而与翻译后GroEL/ES系统共表达会导致动力学不相容，导致大量聚集和活性降低（365.6 U/mg）。结构分析表明，最佳活性与构象灵活性相关，而不是静态结构，这是TF赋予的特征。因此，优化时间动态和伴侣协调对于生产复杂蛋白质至关重要。MMC为解剖这些折叠通路提供了一个强大的平台；在氧化还原工程的大肠杆菌SHuffle菌株中，这种正确的折叠被协同增强，显著提高了可溶性产量（达到450.2 mg/L），同时保持了高比活性（857 U/mg），并证实了氧化细胞质是高效生产的关键。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biochemical Engineering Journal 工程技术-工程：化工

CiteScore

7.10

自引率

5.10%

发文量

380

审稿时长

34 days

期刊介绍： The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology. The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields: Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics Biosensors and Biodevices including biofabrication and novel fuel cell development Bioseparations including scale-up and protein refolding/renaturation Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells Bioreactor Systems including characterization, optimization and scale-up Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis Protein Engineering including enzyme engineering and directed evolution.