重组蛋白SRT-ELP36在大肠杆菌中高效表达的工艺优化研究

Optimization of the High-Level Expression Process of Recombinant Protein SRT-ELP36 in Escherichia coli

摘要

类弹性 - 鱿鱼环齿重组蛋白 (SRT-ELP36) 融合了类弹性蛋白 (ELP) 与鱿鱼环齿蛋白 (SRT) 的序列优势，断裂强度 (630 MPa) 与韧性 (130 MJ/m³) 均优于蛛丝蛋白，在生物医学工程、高性能环保纤维、生物基结构材料等领域具有重要应用潜力。本研究以大肠杆菌 BL21 (DE3) 为表达宿主，通过高密度发酵工艺优化提升 SRT-ELP36 的产量。经单因素实验及 Plackett-Burman (PB) 组合实验，筛选出影响发酵产量的关键因子为酵母提取物、蛋白胨、玉米浆干粉；次要因子为甜菜碱和脯氨酸，一般因子为硫酸铵、硫酸镁、甘氨酸和氯化钾。结合中心复合设计 (CCD) 确定最优补料配方为：酵母提取物 6.8 g/L、蛋白胨 7.5 g/L、玉米浆干粉 8.0 g/L。最后，在 50 L 发酵罐中水平验证优化工艺，通过溶氧与残糖的精准调控实现菌体高密度培养，菌体 OD₆₀₀ 可达 100.2，SRT-ELP36 蛋白的产量最高达到 2.52 g/L，较优化前提高了 33.3%。本研究明确了 SRT-ELP36 高密度发酵的关键营养调控因子及权重，建立了可放大的补料分批发酵工艺，为该重组蛋白的工业化生产提供了工艺支撑。

Abstract

The recombinant elastin-like/squid ring tooth protein SRT-ELP36 integrates the sequence advantages of elastin-like polypeptides and squid ring tooth proteins, exhibiting superior breaking strength and toughness compared to spider silk proteins, and holds great application potential in the field of biomaterials. In this study, Escherichia coli BL21(DE3) was used as the expression host to improve the production of SRT-ELP36 through the optimization of high-density fermentation process. Firstly, single-factor experiments were conducted to screen the nutritional components affecting the expression of SRT-ELP36, and then Plackett-Burman (PB) experiment was used to identify yeast extract, peptone and corn steep liquor powder as the key factors affecting fermentation yield. The weight of each factor was clarified by principal component analysis: the key factors were yeast extract, tryptone and corn steep liquor powder, the secondary key factors were betaine and proline, and the general factors were ammonium sulfate, magnesium sulfate, glycine and potassium chloride. Furthermore, the central composite design (CCD) was utilized to determine the optimal fed-batch medium formulation: 6.8 g/L yeast extract, 7.5 g/L tryptone and 8.0 g/L corn steep liquor powder. Finally, the optimized process was verified in a 50 L bioreactor with the core regulation strategy of optimized fed-batch medium combined with precise control of dissolved oxygen and residual sugar. The results showed that the high-density culture ofE. coli was achieved with OD₆₀₀ reaching 100.2, and the volumetric production of SRT-ELP36 reached 2.52 g/L, which was a 33.3% increase compared with that before optimization. SRT-ELP36 existed as inclusion bodies in the bacterial precipitate, and a standardized SDS-PAGE semi-quantitative detection method was established. This study clarified the key nutritional regulation factors and their weight relationship for the high-density fermentation of SRT-ELP36, and established a scalable fed-batch fermentation process, which laid a technical foundation for the industrial production of SRT-ELP36 recombinant protein.

关键词

大肠杆菌 / 类弹性-鱿鱼环齿重组蛋白 / 响应面法 / 工艺优化 / 高密度发酵

Key words

Escherichia coli / recombinant protein / response surface methodology / process optimization / high-density fermentation

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