Open AccessPreparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli
Author(s) -
Palmer Ira,
Wingfield Paul T.
Publication year - 2012
Publication title -
current protocols in protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.409
H-Index - 32
eISSN - 1934-3663
pISSN - 1934-3655
DOI - 10.1002/0471140864.ps0603s70
Subject(s) - periplasmic space , guanidine , inclusion bodies , escherichia coli , chemistry , centrifugation , cytoplasm , chromatography , size exclusion chromatography , protein purification , cell disruption , biochemistry , recombinant dna , extraction (chemistry) , enzyme , gene
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; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low‐speed centrifugation. Following pre‐extaction (or washing), protein is extracted from washed pellets using guanidine⋅HCl. The solubilized and unfolded protein is either directly folded or further purified by gel filtration in the presence of guanidine⋅HCl as described in this unit. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS‐PAGE. Curr. Protoc. Protein Sci . 70:6.3.1‐6.3.20. © 2012 by John Wiley & Sons, Inc.