Thermosensitive poly(N‐isopropylacrylamide) hydrogel for refolding of recombinant bovine prethrombin‐2 from E. coli inclusion bodies

Abstract

AbstractThe poly(N‐isopropylacrylamide) (PNIPA) hydrogel, which is a kind of temperature‐sensitive polymer, was synthesized by inverse suspension polymerization. The microscopy and scan electron microscopy (SEM) of PNIPA hydrogel were studied. The microscope photograph showed that the particles were in the range of 0.2–0.5mm in diameter, with numerous conjoint pores about 1–2μm spreading all over the surface of the beads. The swelling properties of PNIPA gel beads indicated that the lower critical solution temperature (LCST) of the gel was 33°C. The PNIPA prepared was applied to the renaturation of bovine prethrombin‐2 (pThr‐2) from inclusion bodies produced in E. coli. It was observed that PNIPA was quite efficient in assisting protein renaturation at high protein concentration. When mixing with 105mg/mL PNIPA hydrogel during the refolding, the total activity of the thrombin was about 6222U/mL, compared with only 2800U/mL by simple dilution refolding. The kinetics of pThr‐2 refolding with the absence or the presence of PNIPA was also studied respectively. The time required for the refolding with PNIPA gel was a little bit longer than that by the dilution method owing to the diffusion resistance of the protein into the network of the gel and the hydrophobic interaction between the protein and the polymer. The mechanism of the enhancement for the PNIPA gel to the refolding was further discussed. The porosity of the PNIPA hydrogel allows penetration of the unfolded protein into the inside of the polymer with a hydrophobic side chain, which can facilitate the formation of intermediate via hydrophobic interaction with the unfolded protein and the folding intermediate that are liable to re‐aggregation. About 1.2mg of purified active thrombin could be recovered from 1 L of cells, which greatly facilitated the scale‐up to the quantities of protein necessary for further functional and structural studies. A novel protein renaturation method mediated by PNIPA hydrogel beads, which highly increases the refolding efficiency with easy handling, recycling, and low cost, was proposed. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1734–1740, 2005