Protein refolding for industrial processes

protein inclusion body

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Protein refolding for industrial processesEliana De Bernardez Clark

Inclusion body refolding processes are poised to play a majorrole in the production of recombinant proteins. Improvingrenaturation yields by minimizing aggregation and reducingchemical costs are key to the industrial implementation ofthese processes. Recent developments include solubilizationmethods that do not rely on high denaturant concentrationsand the use of high hydrostatic pressure for simultaneoussolubilization and renaturation.

Addresses

Department of Chemical and Biological Engineering, Tufts University,Medford, MA 02155, USA; e-mail: eliana.clark@tufts.eduCurrent Opinion in Biotechnology2001, 12:202–2070958-1669/01/$ — see front matter

© 2001 Elsevier Science Ltd. All rights reserved.AbbreviationsCTABn-cetyl trimethylammonium bromideDTEdithioerythritolDTTdithiothreitol

GdmClguanidinium chloride

PDGFplatelet-derived growth factorSDSsodium dodecyl sulfateSECsize-exclusion chromatography

Introduction

The need for the efficient production of genetically engi-neered proteins has grown and will continue to grow as aconsequence of the success of the human genome project. Avariety of hosts may be used to produce these proteins, withexpression in bacteria poised to play a major role, particular-ly when the biological activity of the protein product is notdependent on post-translational modifications. Expressionof genetically engineered proteins in bacteria often resultsin the accumulation of the protein product in inactive insol-uble deposits inside the cells, called inclusion bodies. Facedwith the prospect of producing an insoluble and inactiveprotein, researchers usually attempt to improve solubility bya variety of means, such as growing the cells at lower tem-peratures, co-expressing the protein of interest withchaperones and foldases and using solubilizing fusion part-ners, among others [1]. However, expressing a protein ininclusion body form can be advantageous. Large amounts ofhighly enriched proteins can be expressed as inclusion bod-ies. Trapped in insoluble aggregates, these proteins are forthe most part protected from proteolytic degradation. If theprotein of interest is toxic or lethal to the host cell, theninclusion body expression may be the best available pro-duction method. The challenge is to take advantage of thehigh expression levels of inclusion body proteins by beingable to convert inactive and misfolded inclusion body proteins into soluble bioactive products[2–5].

The recent literature includes many examples of therefolding of genetically engineered proteins. A significant

number of these publications deal with the expression

and purification of small amounts of proteins for structure/function relationship and biophysical characterizationstudies. Although valuable, the processes described inthese publications are usually inefficient, include multipleunnecessary steps and have very low recovery yields. Asecond significant fraction of the refolding literature dealswith understanding the folding pathway of a variety of pro-teins and, in particular, early folding events. These studiesare performed with purified proteins that are subjected tounfolding under a variety of conditions, followed by carefully designed and monitored refolding experiments.A third fraction of the refolding literature, and the focus ofthis review, deals with the development of more efficientrefolding methods that can be used for the commercialproduction of genetically engineered proteins

The general strategy used to recover active protein frominclusion bodies involves three steps: inclusion body isola-tion and washing; solubilization of the aggregated protein;and refolding of the solubilized protein (Figure1a).Although the efficiency of the first two steps can be rela-tively high, renaturation yields may be limited by theaccumulation of inactive misfolded species as well as aggre-gates. Because the majority of industrially relevant proteinscontain one or more disulfide bonds, this review focuses onrecent advances in oxidative protein refolding, that is,refolding with concomitant disulfide-bond formation.

Inclusion body isolation, purification andsolubilization

Inclusion bodies are dense, amorphous protein deposits thatcan be found in both the cytoplasmic and periplasmic spaceof bacteria [1,6 ]. Structural characterization studies usingATR-FTIR (attenuated total reflectance Fourier-trans-formed infrared spectroscopy) have shown that theinsoluble nature of inclusion bodies may be due to theirincreased levels of non-native intermolecular β-sheet con-tent compared with native and salt-precipitated protein[7,8]. Cells containing inclusion bodies are usually disruptedby high-pressure homogenization or a combination ofmechanical, chemical and enzymatic methods [6 ,9 ]. Theresulting suspension is treated by either low-speed centrifu-gation or filtration to remove soluble proteins from theparticulate containing the inclusion bodies. The most difficult to remove contaminants of inclusion body proteinpreparations are membrane-associated proteins that arereleased upon cell breakage. Washing steps are performed toremove membrane proteins and other contaminants.Methods used to solubilize prokaryotic membrane proteinscan be adapted to wash inclusion bodies. The most commonwashing steps utilize EDTA, and low concentrations ofdenaturants and/or weak detergents such as Triton X-100,deoxycholate and octylglucoside[6 ,9 ,10,11 ,12,13,P1,P2].

protein inclusion body

Batas, Schiraldi and Chaudhuri [10] recently comparedcentrifugation and membrane filtration for the recoveryand washing of inclusion bodies. Two membrane pore sizes(0.1 and 0.45µm) were compared; the larger pore sizemembrane gave better solvent flux and protein purity.Centrifugation resulted in higher protein purity, probabl …… 此处隐藏：31662字，全部文档内容请下载后查看。喜欢就下载吧 ……