Overview of the purification of recombinant proteins produced in Escherichia coli.
The updated version of this unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. coli. The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a discussion of common problems associated with bacterial protein expression. A flow chart summarizes approaches for establishing solubility and localization of bacterially produced proteins. Purification strategies for both soluble and insoluble proteins are also reviewed. A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. Finally, protein handling, scale and aims of purification, and specialized equipment needed for recombinant protein purification and characterization are discussed. The methodologies and approaches described here are essentially suitable for laboratory-scale operations.
Wingfield
Paul T
PT
National Institutes of Health, Bethesda, Maryland.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D019716:Tissue Inhibitor of Metalloproteinase-2; D017228:Hepatocyte Growth Factor; D008715:Methionine