Solubilization, Folding, and Purification of a Recombinant Peptidoglycan‐Associated Lipoprotein (PAL) Expressed in Escherichia coli. (16th April 2018)

• Record Type: Journal Article
• Authors: Santos, Clelton A.; Souza, Anete P.
• Editors: Coligan, John E.; Dunn, Ben M.; Speicher, David W.; Wingfield, Paul T.
• Is Part Of: Current protocols in protein science. Volume 92(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-04-16
• Subjects: inclusion bodies -- outer membrane protein -- PAL -- protein refolding -- protein solubilization -- protein purification -- recombinant protein; Proteins -- Laboratory manuals; Proteins; Clinical Laboratory Techniques; Genetic Techniques; Immunologic Techniques; Proteins; Laboratory manuals; 572.6028
• DOI: 10.1002/cpps.53
• Languages: English
• ISSNs: 1934-3655

Abstract

Abstract: Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan‐associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram‐negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production. This protocol describes an efficient protein refolding method to solubilize and purify a recombinant PAL. Initially, recombinant PAL–enriched inclusion bodies obtained after the induction of PAL expression in Escherichia coli are treated with 8 M urea and then undergo buffer exchange via dialysis. Afterward, the soluble, recombinant PAL is purified using standard chromatographic methods. © 2018 by John Wiley & Sons, Inc.