Refolding of recombinant proteins

Department of Chemical Engineering, Tufts University, Medford, MA 02155, USA
Current Opinion in Biotechnology (Impact Factor: 8.04). 05/1998; DOI: 10.1016/S0958-1669(98)80109-2

ABSTRACT Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion body protein can be successfully refolded. Aggregation is the leading cause of decreased refolding yields. Developments during the past year have advanced our understanding of the mechanism of aggregation in in vitro protein folding. New additives to prevent aggregation have been added to a growing list. A wealth of literature on the role of chaperones and foldases in in vivo protein folding has triggered the development of new additives and processes that mimic chaperone activity vitro.

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    ABSTRACT: Inclusion bodies were solubilized in a µ-scale system using shaking microtiter plates or a stirred tank reactor in a laboratory setting. Characteristic dimensionless numbers for mixing, the Phase number Ph and Reynolds number Re did not correlate with the kinetics and equilibrium of protein solubilization. The solubilization kinetics was independent of the mixing system, stirring or shaking rate, shaking diameter, and energy input. Good agreement was observed between the solubilization kinetics and yield on the µ-scale and laboratory setting. We show that the inclusion body solubilization process is controlled predominantly by pore diffusion. Thus, for the process it is sufficient to keep the inclusion bodies homogeneously suspended, and additional power input will not improve the process. The high throughput system developed on the µ-scale can predict solubilization in stirred reactors up to a factor of 500 and can therefore be used to determine optimal solubilization conditions on laboratory and industrial scale. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 01/2014; 111(1). DOI:10.1002/bit.24998 · 4.16 Impact Factor
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    ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.
    Applied Microbiology and Biotechnology 12/2012; 97(16). DOI:10.1007/s00253-012-4604-0 · 3.81 Impact Factor
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    ABSTRACT: Of the various protein refolding methods, direct dilution is one of the simplest and easiest for scaling up the refolding process. However, it requires a large amount of refolding buffer, often utilizes a number of chemicals, and results in a low final protein concentration. In this report, we demonstrate that reduced dithiothreitol (DTTred), a carryover from denaturation, is a crucial and adverse factor in lysozyme refolding. Accordingly, we proposed a method of using high concentration of oxidized glutathione (GSSG) in the refolding buffer to eliminate excess DTTred and aid in the refolding of lysozyme. The efficiency of this method is 84%, which resulted in a high final refolded protein concentration of 1.5 g/l and required only a low dilution factor (4×). Furthermore, compared with the traditional 50× direct dilution (resulting in a similar yield of 74%), the low dilution factor required much less GSSG and other constituents.
    PROCESS BIOCHEMISTRY 12/2012; 47(12):1883–1888. DOI:10.1016/j.procbio.2012.06.026 · 2.52 Impact Factor

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