重组人肽抗生素hPAB-β的初步纯化

Preliminary Purification of Recombinant Human Peptide Antibiotic(hPAB-β)

目的 探索重组人肽抗生素hPAB β的纯化工艺 ,为制备高纯度、高活性的重组hPAB β奠定基础。方法 将构建的含pQE32 CP hPAB β重组质粒的基因工程菌扩大培养 ,诱导表达后所得菌体经溶菌酶法裂解 ,鉴定融合蛋白表达形式 ;以含融合蛋白的溶液作为纯化的初始样品 ,采用Ni NTAresin亲和层析柱对融合蛋白进行纯化 ,用CNBr裂解 ,利用SephadexG 5 0、Bio gelP6DG凝胶进行分子筛层析 ,利用Macro PrepHighS进行阳离子交换层析 ,对层析峰行Tris Tricine电泳分析。结果 融合蛋白以包涵体形式存在 ;亲和层析获得了纯度为 82 .6 %的融合蛋白 ,CNBr作用 2 0h可完成融合蛋白的裂解 ;目的肽经纯化后 ,纯度达 95 %以上。结论 已初步建立了重组人肽抗生素hPAB β的纯化工艺 ,并获得了高纯度的肽抗生素hPAB β。

Objective To explore the purification procedure of recombinant human peptide antibiotic(rhPAB β)and lay a foundation of preparing rhPAB β with high purity and activity.Methods Construct recombinant plasmid pQE32 CP hPAB β,transform to engineering bacterial strain and express under the induction of IPTG.Lyze the induced bacteria by lysozyme,identify the expressing form of the target fusion protein,then purify the fusion profein by Ni NTA resin affinity chromatography.Lyze the purified product with CNBr and further purify by Sephadex G 50 and Bio gel P6 DG molecular sieve chromatographies,as well as Macro Prep High S cation exchange chromatography.Analyze the purified product by Tris Tricine electrophoresis. Results The expressed fusion protein existed in inclusion body form,and its purity was 82.6% after affinity chromatography.It was completely lyzed after treatment with CNBr for 20 h and reached a purity of above 95% after further purification.Conclusion A purification procedure of rhPAB β was preliminarily developed,and highly purified rhPAB β was obtained.