Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli.
Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications.
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; Division of General Internal Medicine, Department of Medicine, University of Alberta, Edmonton, AB, Canada. Electronic address: pmhwang@gmail.com.
Peter M
PM
Hwang
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Jonathan S
JS
Pan
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Brian D
BD
Sykes
D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics*; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins
Chemical cleavage; Fusion protein; Inclusion body