Folding and purification of insoluble (inclusion body) proteins from Escherichia coli

Current Protocols in Protein Science
Paul T WingfieldS M Liang

Abstract

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed and the are aggregates, (2) the cell wall and outer membrane components of the aggregates are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein which is then partially purified by gel filtration before folding, and then the protein is folded and oxidized by simple dialyzed against water. A Support Protocol is included for r...Continue Reading

References

Apr 1, 1979·Analytical Biochemistry·P W RiddlesB Zerner
Jan 1, 1975·International Journal of Peptide and Protein Research·L GráfT A Bewley
Sep 29, 1992·Biochemistry·S Bastiras, J C Wallace
Jul 17, 1992·Science·J F Bazan
Jul 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·P A Sherman, J A Fyfe
Jan 1, 1990·Methods in Enzymology·C M Stoscheck
Jan 1, 1989·The International Journal of Biochemistry·T Vanha-Perttula, J Kasurinen
Oct 1, 1989·Journal of Protein Chemistry·B N ViolandL A Bentle
Sep 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·S S Abdel-MeguidL A Bentle
Aug 1, 1986·Journal of Medicinal Chemistry·J S NewD L Temple
May 15, 1986·Analytical Biochemistry·J L BrowningJ E Smart
Jul 1, 1987·The Biochemical Journal·M P Weir, J Sparks
Sep 1, 1986·International Journal of Peptide and Protein Research·C SecchiS Ronchi
Jul 31, 1985·Biochemical and Biophysical Research Communications·K KatoA Kakinuma
Jul 15, 1985·The Biochemical Journal·S M LiangD R Thatcher
Jan 1, 1970·Advances in Protein Chemistry·G K Ackers
Jul 11, 1983·Nucleic Acids Research·R DevosW Fiers
Oct 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·R J RobbV Chowdhry
Jan 1, 1984·Methods in Enzymology·D B Wetlaufer
Apr 1, 1984·Analytical Biochemistry·T W ThannhauserH A Scheraga
Jan 1, 1994·Annual Review of Biochemistry·R A Katz, A M Skalka
Apr 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·F D BushmanR Craigie
Jan 1, 1993·Annual Review of Immunology·Y MinamiT Taniguchi
Jan 1, 1960·Methods of Biochemical Analysis·P TALALAY
Jul 17, 1992·Science·D B McKay

❮ Previous
Next ❯

Citations

Mar 25, 2016·Biochemistry·Gavin Ray OwenNichole Michelle Cerutti
Apr 23, 2008·Current Protocols in Neuroscience·K J Petty
Aug 29, 1998·The Journal of Biological Chemistry·L WorthS Clark
Dec 13, 2002·The Journal of Biological Chemistry·Inken G HuttnerJeremy Thorner
Feb 25, 2020·Biotechnology and Applied Biochemistry·Reyhane KeshavarzAli Asghar Deldar

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.