III型类人胶原蛋白在大肠杆菌重组表达及发酵制备

Recombinant expression and fermentation of type III human-like collagen in Escherichia coli

摘要:

【背景】胶原蛋白广泛应用于日用化工及生物医药中，相比传统方法，基因工程方法制备胶原蛋白具有避免病毒隐患、产量高等优点，逐步受到广泛关注。【目的】获得III型类人胶原蛋白基因，实现大肠杆菌中的异源表达。【方法】以人III型胶原蛋白α1链为模板，(Gly-X-Y)为最小研究单位，优选亲水性氨基酸，设计目标基因kit，构建重组大肠杆菌(Escherichia coli) pET-28a(+)-kit/BL21(DE3)，并对其结构进行表征。【结果】类人胶原蛋白基因kit成功在大肠杆菌体系中表达，表达量约为0.53 g/L，7 L发酵罐上补料发酵后其最大表达量提高至3.02 g/L，亲和层析纯化类人胶原蛋白纯度约为91%，对其进行N端测序、氨基酸分析、质谱分析及圆二色谱分析，确定类人胶原蛋白成功表达。【结论】类人胶原蛋白的成功表达为未来规模化制备及其在日用化工及生物医药行业的应用奠定了基础。

Abstract:

[Background] Collagen is widely used in daily-use chemical industry and biomedicine. Genetic engineering method for preparing collagen exhibits several advantages, such as avoiding virus infection and high expression level, which has attracted substantial attention in recent years. [Objective] A human-like collagen gene was obtained to achieve heterologous expression in E. coli. [Methods] Using human type III collagen α1 chain as template and (Gly-X-Y) as the smallest research unit, preferably selecting hydrophilic amino acids, designing and synthesizing gene kit, and constructing recombinant E. coli pET-28a(+)-kit/BL21(DE3), and characterizing it. [Results] The human-like collagen gene kit was successfully expressed in E. coli with expression level of about 0.53 g/L. After fed-batch fermentation on a 7 L fermenter, the maximum expression level was increased to 3.02 g/L. Purification by affinity chromatography was performed, which obtained the human-like collagen with a purity of about 91%. Human-like collagen was analyzed by N-terminal sequencing, amino acid analysis, mass spectrometry, and circular dichroism analysis to determine the successful expression of human-like collagen. [Conclusion] The successful expression of human-like collagen would lay the foundation for future preparation and its practical application in the daily chemical and biomedical industries.