Refolding of recombinant proteins

Department of Chemical Engineering, Tufts University, Medford, MA 02155, USA
Current Opinion in Biotechnology (Impact Factor: 7.12). 05/1998; 9(2):157-163. DOI: 10.1016/S0958-1669(98)80109-2


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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    • "These refolding techniques have some disadvantages, such as that the dialysis procedure requires large amounts of reagents, long treatment times (24), and can also cause the adhesion of protein to the membranes used (25). Occasionally protein aggregates formed (26). The disadvantages of the dilution method are the large processing volumes involved, higher costs, and the “step-change” in denaturant concentration to native conditions may result in aggregation (27). "
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    ABSTRACT: Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10-8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10-7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.
    Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 06/2014; DOI:10.1590/1414-431X20143276 · 1.01 Impact Factor
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    • "When proteins are purified from IB, urea-denatured and then refolded in vitro, addition of osmolytes (also called chemical chaperones) in the 0.1–1 M range of concentration increases the yield of soluble protein (Rudolph and Lilie, 1996; Clark, 1998; Tsumoto et al., 2003; Alibolandi and Mirzahoseini, 2011). This situation can be mimicked in vivo by supplementing the culture media with osmolytes such as proline, glycine-betaine, and trehalose (de Marco et al., 2005). "
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    ABSTRACT: Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.
    Frontiers in Microbiology 04/2014; 5:172. DOI:10.3389/fmicb.2014.00172 · 3.99 Impact Factor
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