Article

Refolding of recombinant proteins

Department of Chemical Engineering, Tufts University, Medford, MA 02155, USA
Current Opinion in Biotechnology (Impact Factor: 8.04). 05/1998; 9(2):157-163. 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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    • "Recombinant proteins can be produced efficiently in Escherichia coli as inclusion bodies (IBs). For recovery of the active product, these dense particles of aggregated protein have to undergo different processing steps such as recovery of IBs by cell lysis, fractionation of cell lysates and removal of contaminants and subsequent solubilization and refolding of the IBs (Clark, 1998; Dürauer et al., 2009; Eiberle and Jungbauer, 2010; Freydell et al., 2007; Georgiou and Valax, 1999; Jungbauer and Kaar, 2007). A crucial step during IB processing is efficient solubilization of the protein (Kim and Lee, 2000; Singh and Panda, 2005). "
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    ABSTRACT: Screening for optimal refolding conditions for recombinant protein overexpressed in Escherichia coli as inclusion bodies is often carried out on micro-scale in non-agitated reactors. Currently, scale up of refolding of Npro fusion proteins is based on geometric similarity and constant Re number. Refolding/cleavage kinetics is recorded offline by HPLC and via fluorescence intensity. We show that the results for refolding obtained on the micro-scale can be transferred to the laboratory scale stirred tank reactor, with increases in scale up to a factor of 5000, with high agreement of kinetic constants and yield. Progress of refolding kinetics on the laboratory scale is monitored inline by attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR). Addressing the demands for better process understanding, we demonstrate that ATR-FTIR enables the inline monitoring of refolding processes on the laboratory scale, replacing offline analysis which delivers the results with a time delay. Implementing inline monitoring will allow the integration of process control, thereby resulting in a more efficient and knowledge based production process.
    PROCESS BIOCHEMISTRY 07/2014; 49(7). DOI:10.1016/j.procbio.2014.03.022 · 2.52 Impact Factor
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    • "Recombinant proteins can be produced efficiently in Escherichia coli as inclusion bodies (IBs). For recovery of the active product, these dense particles of aggregated protein have to undergo different processing steps such as recovery of IBs by cell lysis, fractionation of cell lysates and removal of contaminants and subsequent solubilization and refolding of the IBs (Clark, 1998; Dürauer et al., 2009; Eiberle and Jungbauer, 2010; Freydell et al., 2007; Georgiou and Valax, 1999; Jungbauer and Kaar, 2007). A crucial step during IB processing is efficient solubilization of the protein (Kim and Lee, 2000; Singh and Panda, 2005). "
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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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    • "In addition to their functional use for antigen targeting, the V H /V L construct presents substantial benefits from a fundamental design perspective as the F V region can serve as a model system for understanding the folding behavior of multi-domain proteins. However, intra-or inter-protein interactions can cause misfolding and aggregation that serve as kinetic and/or thermodynamic limitations during the process of protein (re)folding [5] [6] [7]. The folding process is further complicated for engineered proteins that often consist of multiple domains with highly disparate stabilities and folding propensities, in that they often must be folded in vitro without the assistance of molecular chaperones. "
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    ABSTRACT: Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (VH/VL) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and VH/VL domains had considerably different unfolding free energies (ΔGUNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the VH/VL folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the VH/VL domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the VH/VL domain played a key role in aggregation of this antibody fusion protein.
    Biochemical Engineering Journal 12/2013; 81:8–14. DOI:10.1016/j.bej.2013.09.015 · 2.37 Impact Factor
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