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Review
. 2014 Feb 20;4(1):235-51.
doi: 10.3390/biom4010235.

Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies

Hiroshi Yamaguchi  1 , Masaya Miyazaki  2
Affiliations
Review

Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies

Hiroshi Yamaguchi et al. Biomolecules. .

Abstract

Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

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Figures

Figure 1
Figure 1
Schematic illustration of the two types of dialysis method for the removal of denaturants from denatured (solubilized) protein solutions. In one-step dialysis, the denaturant concentration around the protein rapidly decreases through diffusion, leading to aggregates. In step-wise dialysis, gradual removal of denaturant from the denatured proteins occurs.
Figure 2
Figure 2
Typical chemical additives used for protein refolding. (a) Denaturants; (b) Inhibitors; (c) Stabilizers.
Figure 3
Figure 3
Models of the interactions between the surface of the protein with water and chemical additives. The additives and molecular water are represented by colored circles and ellipses, respectively: denaturant (strong chaotropic reagent), red; inhibitor (moderate chaotropic reagent), dark blue; stabilizer (osmotic reagent), green; and water, light blue.
Figure 4
Figure 4
Microfluidic chip used for protein refolding [15]. (a) Designed microfluidic chips. In MR1, the denaturant concentration around the protein rapidly decreases because of diffusion, which is expected to have a similar mechanism to one-step dialysis and dilution. In MR2, the denaturant concentration shows a step-wise decrease, which is a similar mechanism to step-wise dialysis. The denatured protein was injected into channel a. The diluting buffer was injected into channels b and c; (b) Confocal fluorescence microscope image at the junction in MR1 showing laminar flow of the urea stream through the diluting buffer streams. The focused urea stream contains N-(4-nitrobenzo-2-oxa-1,3-diazolyl)amine (NBD) as an indicator; (c) Relative fluorescence intensities of NBD in the urea stream as a function of the distance from the inlet (position a). Flow rates (μL/min) are shown in the graph.
Figure 5
Figure 5
Citrate synthase (CS) refolding by microfluidic chips [15]. (a) The recovered enzymatic activities of CS using different refolding approaches. Flow rates for MR1: channel a (denatured CS), 10 μL/min; and channel b (buffer), 90 μL/min. Flow rates for MR2: channel a (denatured CS), 10 μL/min; channel b (buffer), 10 μL/min; and channel c (buffer), 80 μL/min. Folded CS was prepared by dialysis. Unfolded CS was assayed in 2.5 M urea. The diluted sample was directly diluted by buffer in a test tube; (b) The effect of the different flow rates of the diluting buffers (channels b and c) on CS refolding. Relative fluorescence intensities of NBD in the urea stream at the junctions in MR2; (c) The recovered enzymatic activities of refolded CS in MR2. Flow rates of channels a, b, and c (μL/min) are shown in the graph.
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References

    1. Swartz J.R. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 2001;12:195–201. doi: 10.1016/S0958-1669(00)00199-3. - DOI - PubMed
    1. Clark E.D.B. Protein refolding for industrial processes. Curr. Opin. Biotechnol. 2001;12:202–207. doi: 10.1016/S0958-1669(00)00200-7. - DOI - PubMed
    1. Baneyx F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999;10:411–421. doi: 10.1016/S0958-1669(99)00003-8. - DOI - PubMed
    1. Baneyx F., Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 2004;22:1399–1408. doi: 10.1038/nbt1029. - DOI - PubMed
    1. Prasad S., Khadatare P.B., Roy I. Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 2011;77:4603–4609. doi: 10.1128/AEM.05259-11. - DOI - PMC - PubMed

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