重组弹性蛋白表达与发酵工艺优化及放大

Optimization and scale-up of recombinant elastomeric protein expression and fermentation process

摘要:

重组弹性蛋白因优异的生物相容性、可调控的机械性能及仿生微环境特性，在生物医药与食品领域具有广阔应用前景，但生产效率低下制约了其产业化。为解决这一问题，本研究通过多阶段优化策略，建立了稳定高效的规模化制备工艺。首先，对ure(80)进行密码子优化，产量提高至0.42 g/L。通过靶向截短分析，定位第50-59位氨基酸为限制表达的关键区域，进而对该区域进行二次密码子与mRNA结构优化，并结合核糖体结合位点(ribosome binding site, RBS)理性设计，构建高产工程菌株大肠杆菌(Escherichia coli) BL21(DE3)/pET-28a-80opt-R1，产量达0.62 g/L，较初始提高2倍。其次，采用无机盐培养基，通过响应面法优化碳源、氮源、微量元素及维生素B1等组分，同时优化诱导温度、时间、pH及IPTG浓度等发酵参数，使蛋白产量提升至3.5 g/L。经发酵罐放大，利用其优异的传质与控制能力，最终产量达5.0 g/L，较初始产量(0.3 g/L)提高16.7倍。细胞功能验证表明，增殖与黏附能力未受影响。本研究为重组弹性蛋白的规模化制备提供了可靠工艺，也为其他复杂蛋白的产业化提供了参考路径。

Abstract:

Recombinant elastomeric protein exhibits excellent biocompatibility, tunable mechanical properties, and biomimetic microenvironment characteristics, offering broad application prospects in the biomedical and food sectors. However, its low production efficiency has hindered further research and industrial translation. To address this challenge, this study established a stable and efficient large-scale preparation process through a multi-stage optimization strategy. First, codon optimization of ure(80) increased the yield to 0.42 g/L. Targeted truncation analysis identified amino acid residues 50-59 as a key region limiting expression. Subsequent secondary codon optimization and mRNA structure optimization targeting this region, combined with rational design of the ribosome-binding site (RBS), led to the construction of a high-producing engineered strain, Escherichia coli BL21(DE3)/pET-28a-80opt-R1, which achieved a yield of 0.62 g/L—a two-fold increase compared with the initial yield. Second, response surface methodology was employed to optimize the concentrations of carbon source, nitrogen source, trace elements, and vitamin B1 in a minimal salt medium, while fermentation parameters including induction temperature, induction duration, pH, and IPTG concentration were optimized, increasing the protein yield to 3.5 g/L. Upon scale-up in a fermenter, leveraging its superior mass transfer and process control capabilities, the final yield reached 5.0 g/L, representing a 16.7-fold increase over the initial yield (0.3 g/L). Cellular functional assays confirmed that cell proliferation and adhesion were not adversely affected. This study provides a reliable process for the large-scale preparation of recombinant elastomeric protein and offers a reference for the industrial production of other complex proteins.