Enhanced protein renaturation by temperature-responsive polymers

Abstract

The application of temperature‐sensitive polymer (PNIPAAm) for the renaturation of β‐lactamase from inclusion bodies was investigated. It was observed that PNIPAAm was more effective than PEG in enhancing protein renaturation. At a concentration of 0.1%, PNIPAAm improved the yield of β‐lactamase activity by 41% from 46.5 to 65.4 IU/mL, compared to 26% with PEG from 46.5 to 58.7 IU/mL. Kinetic study indicated that PNIPAAm did not significantly affect the initial rate of protein renaturation but did increase final activity yield. In the presence of PEG and PNIPAAm, the activity yields increased with temperature, indicating that hydrophobic interactions between denatured protein and polymer molecules contributed to the enhanced protein renaturation with polymers. The sequential addition approach, aiming at enhancing protein renaturation by reducing local protein concentration during renaturation, was also shown effective in enhancing protein renaturation, especially in the presence of polymers. With the sequential addition approach, the activity yield was increased by 60.5% from 46.5 to 74.6 IU/mL with PNIPAAm. Similar behavior was also observed with PEG. PNIPAAm exhibited similar behavior as PEG on the renaturation of β‐lactamase in terms of temperature effect and concentration effect, indicating that the mechanism for enhanced protein renaturation for the two polymers might be similar. PNIPAAm exhibits a lower critical solution temperature (LCST) of 32°C and can be effectively separated from aqueous solution and recycled. A protein renaturation process employing PNIPAAm, which offers the advantages of enhanced renaturation efficiency, minimum loss of protein aggregates, and ease of polymers recycling, was proposed. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 505–512, 2000.

Refolding of recombinant N-acetyl-d-glucosamine 2-epimerase by a fed-batch process

“…The cell pellets harvested were re-suspended in 10 mL lysis buffer containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and 2 g/mL of lysozyme at pH 8.0, and incubated at room temperature for 15 min before sonication (Lin et al, 2000). The soluble fraction and the insoluble fraction of the ruptured cells were separated by centrifugation at 10,000 Â g for 10 min.…”

“…For example, at similar initial protein concentrations, a recovery yield of ca. 75% have been reported for b-lactamase and carbonic anhydrase, respectively (Chen et al, 2003;Lin et al, 2000). This result indicates that the solubilized GlcNAc 2-epimerase is highly aggregation-prone.…”

“…The inhibition of protein re-aggregation during protein refolding is believed to be mediated by the hydrophobic interactions between PEG and the aggregation-prone folding intermediate . We have recently found that a temperature-responsive polymer, poly(N-isopropylacrylamide), can also be used for improved protein refolding (Chen et al, 2003;Lin et al, 2000).…”

Interpolyelectrolyte complexes of soybean peroxidase with thermoresponsive copolymers

“…Despite a considerable bulk of studies dealing with the inter polyelectrolyte complexes of proteins [22][23][24][25][26], the systematic data on conformational changes in ther moresponsive polymers and globular proteins result ing from complexation are very scanty. There are almost only single facts derived by indirect methods [27][28][29][30]. An exception is a thermodynamic study devoted to the conformational aspects of complex ation of lysozyme with thermoresponsive copolymers [31].…”

Molecular-assisted refolding: Study of two different ionic forms of recombinant human fibroblast growth factors

“…Other additives, such as sugars (Arakawa and Timasheff, 1982) and glycerol (Gekko and Timasheff, 1981) enhanced refolding yields by stabilizing native proteins. More recently, the use of stimuli-responsive polymers such as temperature and pH-sensitive polymers to guide correct refolding of proteins is gaining increasing attention from the biotechnology community considering the potential ability of these 'smart' polymers to simultaneously refold and purify proteins (Kudou et al, 2003;Lin et al, 2000;Lu et al, 2005;Mondal et al, 2007). Eudragit S-100 is one such polymer that has been reported to improve the refolding yield of chemically denatured ␣-chymotrypsin (Roy and Gupta, 2003).…”