对在大肠杆菌中表达的重组人白细胞介素-1β进行两步纯化。

A two step purification of recombinant human interleukin-1 beta expressed in E. coli.

作者信息

Yem A W, Curry K A, Tomich C S, Deibel M R

机构信息

Biopolymer Chemistry and Molecular Biology, Upjohn Company, Kalamazoo, Michigan 49001.

出版信息

Immunol Invest. 1988 Aug-Oct;17(6-7):551-9. doi: 10.3109/08820138809030588.

DOI:10.3109/08820138809030588

PMID:3069712

Abstract

Recombinant human interleukin-1 beta has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1 beta is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1 beta (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 x 10(7) units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1 beta sera.

摘要

利用从SKhep1RNA获得的cDNA克隆，已在大肠杆菌中高效表达重组人白细胞介素-1β。通过冻融提取，随后在苯基琼脂糖上进行疏水相互作用色谱，将重组白细胞介素-1β纯化至表观均一性。该方法无需使用变性剂，如有需要，也可在不进行柱色谱步骤的情况下，提供纯的重组白细胞介素-1β（每升大肠杆菌培养物可达15毫克）。纯度通过一维聚丙烯酰胺凝胶上的单一条带和二维聚丙烯酰胺凝胶上的单个斑点来定义。在成纤维细胞增殖试验中，纯化后的蛋白质表现出1×10⁷单位/毫克的生物活性，并显示与兔抗人白细胞介素-1β血清发生交叉反应。