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

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.