通过基因工程法优化人胶原蛋白的密码子序列,以人Ⅲ型胶原蛋白α1链为模板,(Gly-X-Y)为最小研究单位,构建重组质粒pET30a(+)-3CH,利用Escherichia coli发酵和蛋白纯化技术制备重组人Ⅲ型胶原蛋白(Recombinant human typeⅢcollagen,RHCⅢ).研究结果证实,RHC Ⅲ的表达量为3.5 g/L,利用亲和/离子交换层析工艺纯化后的RHC Ⅲ纯度为95.44％,RHC Ⅲ蛋白溶液在4 ℃下可以保存12个月,稳定性良好,无细胞毒性,为重组人胶原蛋白产业化奠定了良好基础.
Expression and Purification Process of Recombinant Human Type Ⅲ Collagen in Escherichia Coli
The codon sequence of human collagen was optimized by genetic engineering method.Using the a1 chain of human collagen Ⅲ as the template and(Gly-X-Y)as the minimum research unit,recombinant plasmid PET30a(+)-3CH,Escherichia coli fermentation and protein purification technology were used to prepare recombinant human type Ⅲ collagen(RHC Ⅲ).The results confirmed that the expression level of RHC Ⅲ was about 3.5 g/L,and the purity of RHC Ⅲ was about 95.44％after purification by affinity/ion exchange chromatography.The RHC Ⅲ protein solution could be stored at 4 ℃ for 12 months,with good stability and no cytotoxicity.The results laid a good foundation for the industrialization of recombinant human collagen.
genetic engineeringrecombinant human type Ⅲ collagenEscherichia coliprotein purification
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