Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Abstract

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

Optimizing conditions for production of high levels of soluble recombinant human growth hormone using Taguchi method

“…Glycerol is a chemical chaperone that has been known as protective element for proteins against denaturation by increasing the relative hydration around the proteins. In a study, it has been indicated that using glycerol as a chemical chaperone increased the yield of production of the soluble Human Phenylalanine Hydroxylase Mutant Enzyme [1]. Ethanol not only acts as a chemical chaperone but also as an inducing factor for synthesis of molecular chaperone by bacterial cells.…”

“…Chemical chaperones are low molecular weight compounds including glycerol and ethanol which have been used to prevent the formation of aggregates into inclusion bodies during invitro folding. Glycerol due to the enhancement of hydrophobic interactions can act as a stabilizer of protein conformation [1,12]. Recently, invitro studies in transfected cells demonstrated that glycerol can correct the defective folding of delta F508 CFTR in cystic fibrosis by stabilizing an otherwise unstable intermediate in CFTR biosynthesis [21].…”

Wanted: more monitoring and control during inclusion body processing

“…Subsequently, consecutive IB washing steps are performed, before the IBs are solubilized with denaturants, like urea, sarkosyl or guanidine hydrochloride, and the cell debris is removed by centrifugation. After solubilization, the POI needs to be refolded in its native conformation which is usually achieved by dilution, dialysis or on-column refolding with the aid of small molecule additives (Alibolandi and Mirzahoseini 2011; Ling et al 2015). Finally, the refolded POI might undergo a purification step to further increase purity.…”

Charge-perturbation dynamics — a new avenue towards in silico protein folding

“…Misfolding events can have detrimental and sometimes catastrophic effects on the ability of the protein to perform its function, on its distribution in various cell compartments, and on its recognition by other species 1 . Therefore, the study of protein folding, unfolding, and misfolding is critical to our understanding and manipulation of pathophysiological mechanisms and biotechnological processes 2-4 . Great advances in the field of in silico modelling, including molecular dynamics (MD), have helped understand important aspects of folding, such as the fact that protein-folding energy landscapes are funnel-shaped, or that proteins fold in units of secondary structures 5-7 .…”