大肠杆菌分泌表达重组人肝再生增强因子的纯化、鉴定与活性研究

目的建立一种大肠杆菌分泌表达型重组人肝再生增强因子(rhALR)的纯化工艺,并对产品进行鉴定和活性研究。方法通过沉淀、色谱等方法的条件优化,建立纯化工艺,通过电泳、HPLC、N-末端氨基酸测序、免疫杂交、同位素掺入、全波长扫描等方法对纯化产品进行鉴定和活性测定。结果建立了rhALR的纯化工艺路线,40%(NH4)2SO4沉淀,Butyl-S FF疏水色谱,CM-S FF离子交换色谱,Superdex-75分子筛色谱纯化,重组蛋白质的纯度大于95%。该蛋白质稳定存在的形式为二聚体,其单体的相对分子质量为15 000。该蛋白质结合辅酶黄素腺嘌呤二核苷酸(FAD),对损伤后的肝细胞具有明显的促进作用,明显优于以包涵体形式表达的rhALR。结论从大肠杆菌分泌表达体系能分离纯化得到高纯度、具有生物学功能和活性的rhALR,是潜在的临床治疗肝病药物。

Purification,identification and bioactivity of recombinant human augmenter of liver regeneration secreted expression in E.coli

PurposeTo purify,identify and characterize the recombinant human augmenter of liver regeneration(rhALR) secreted expression in E.coli.MethodsPrecipitation and chromatography were used to purify the recombinant protein.SDS-PAGE,HPLC,sequencing of N-terminus and full wavelength scanning were used to determine the recombinant protein.ResultsPurification process was established starting with precipitation of 40%(NH_4)_2SO_4,followed by hydrophobic chromatography with Butyl-S FF,then ion chromatography with CM-S FF,and finally molecular sieve chromatography with Superdex-75.The purity of rhALR was above 95%. The molecular weight of rhALR is about 15 000 on the reduced SDS-PAGE and 30 000 on the non-reduced SDS-PAGE,indicating rhALR was present as a homodimer in E.coli.The sequence of the N-terminus of rhALR was consistent with the deduced codes from cDNA of hALR.Secreted rhALR is linked with flavin adenine dinucleotide(FAD) and significantly by promoted the DNA synthesis of hepatocytes after acute hepatic injury by CCl_4.ConclusionSecreted rhALR with high purity and bioactivity would be produced through the established purification process using E.coli expression system.It was a potential and efficient source of rhALR in clinical practice for treating hepatic diseases.