Palmer Ira, Wingfield Paul T
National Institutes of Health, Bethesda, Maryland.
Curr Protoc Protein Sci. 2004 Nov;Chapter 6:6.3.1-6.3.18. doi: 10.1002/0471140864.ps0603s38.
High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates and this unit describes preparation of washed pellets and solubilization of the protein using guanidine x HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT or further purified by gel filtration in the presence of guanidine x HCl as idescribed here. A support protocol describes the removal of guanidine x HCl from column fractions so they can be monitored by SDS-PAGE.
许多重组蛋白在大肠杆菌中的高水平表达会导致形成通常称为包涵体的高度聚集蛋白。包涵体通常在细胞质中形成；或者，如果使用分泌载体，它们可以在周质空间中形成。包涵体可以从细胞裂解物中回收，本单元描述了洗涤沉淀的制备以及使用盐酸胍溶解蛋白质的方法。提取的未折叠蛋白可以按照单元中所述直接折叠，或者如本文所述在盐酸胍存在下通过凝胶过滤进一步纯化。一个支持方案描述了从柱级分中去除盐酸胍的方法，以便可以通过SDS-PAGE进行监测。
