Xiang Zhi-Xiang, Gong Jin-Song, Shi Jin-Hao, Liu Chun-Fang, Li Heng, Su Chang, Jiang Min, Xu Zheng-Hong, Shi Jin-Song
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Lihu Avenue No. 1800, Wuxi, 214122, People's Republic of China.
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
Bioresour Bioprocess. 2022 Nov 4;9(1):117. doi: 10.1186/s40643-022-00605-4.
Collagen, the highest content protein in the body, has irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding the recombinant type III human-like collagen α1 chain fragment was integrated into P. pastoris genome after partial amino acids were substituted. Combined with promoter engineering and high-density fermentation technology, soluble secretory expression with the highest yield of 1.05 g L was achieved using two-stage feeding method, and the purity could reach 96% after affinity purification. The determination of N/C-terminal protein sequence were consistent with the theoretical expectation and showed the characteristics of Gly-X-Y repeated short peptide sequence. In amino acid analysis, glycine shared 27.02% and proline 23.92%, which were in accordance with the characteristics of collagen. Ultraviolet spectrum combined with Fourier transform infrared spectroscopy as well as mass spectrometry demonstrated that the target product conformed to the characteristics of collagen spectrums and existed as homologous dimer and trimer in the broth. This work provided a sustainable and economically viable source of the recombinant type III human-like collagen.
胶原蛋白是人体内含量最高的蛋白质，具有不可替代的生物学功能，在食品、美容和医学领域受到广泛关注，市场需求巨大。在对部分氨基酸进行替换后，将编码重组III型人源化胶原蛋白α1链片段的基因整合到巴斯德毕赤酵母基因组中。结合启动子工程和高密度发酵技术，采用两阶段补料法实现了可溶性分泌表达，最高产量达到1.05 g/L，经亲和纯化后纯度可达96%。N/C端蛋白质序列测定结果与理论预期一致，呈现出Gly-X-Y重复短肽序列的特征。氨基酸分析表明，甘氨酸占27.02%，脯氨酸占23.92%，符合胶原蛋白的特征。紫外光谱、傅里叶变换红外光谱以及质谱分析表明，目标产物符合胶原蛋白光谱特征，在发酵液中以同源二聚体和三聚体形式存在。这项工作提供了一种可持续且经济可行的重组III型人源化胶原蛋白来源。
