胶原蛋白（Collagen）目前广泛应用于食品、医疗、美容等领域，然而，重组胶原蛋白存在包涵体形式表达及纯度较低等问题，限制了其规模化生产及制备应用。该研究在I型人源化胶原蛋白hCOL1A1的基础上，在其N末端融合了小分子泛素样修饰蛋白（SUMO）和6×His标签，构建了重组表达载体pET21a（+）-SUMO-hCOL1A1，并转化至大肠杆菌BL21（DE3）。通过对工程菌BL21（DE3）/pET21a（+）-SUMO-hCOL1A1进行IPTG诱导表达、培养条件的优化及蛋白的分离纯化，实现了重组I型人源化胶原蛋白hCOL1A1在大肠杆菌中的可溶性表达及有效纯化。工程菌破碎上清经Ni-NTA亲和层析、TEV酶切、离子交换层析纯化，可收获纯度95%以上的hCOL1A1蛋白。所确定的25 ℃诱导培养温度，添加0.4 mmol/L IPTG的最佳诱导培养条件，进一步在5 L发酵罐进行分批补料发酵放大，最终蛋白产量达到1.43 g/L。该研究实现了hCOL1A1蛋白的可溶表达、5 L发酵罐生产及纯化，为重组胶原蛋白大规模生物合成及纯化提供了参考。

Collagen is currently widely used in the food, medical, and beauty industries. However, isolating recombinant collagens poses considerable challenges because of factors such as inclusion body expression and low purity, limiting its large-scale production and application. Based on humanized type I collagen (hCOL1A1), a recombinant expression vector pET21a(+)-SUMO-hCOL1A1 was constructed by fusing a small ubiquitin-like modifier protein (SUMO) and 6×His tag at the N-terminus of hCOL1A1, which was then transformed into Escherichia coli BL21 (DE3). Soluble expression and purification of recombinant hCOL1A1 in E. coli were achieved through IPTG induction, optimization of the culture conditions, and protein purification of the engineered strain produced by BL21/pET21a(+)-SUMO-hCOL1A1. The supernatant of the engineered strain was purified through Ni-NTA affinity chromatography, TEV enzyme digestion, and ionexchange chromatography, ultimately yielding hCOL1A1 protein with a purity of over 95%. The optimal culture conditions were determined to be an induction temperature of 25 ℃ and a concentration of 0.4 mmol/L IPTG. Further scale-up in 5 L fed-batch fermentation resulted in a final protein yield of 1.43 g/L. Soluble expression of the hCOL1A1 protein, 5 L bioreactor production, and purification were achieved, serving as a valuable reference for the large-scale biosynthesis and purification of recombinant collagen.

国家重点研发计划项目（2021YFC2102700）