Renaturation Of Proteins Assisted By Temperature-sensitive Poly(N-isopropyl Acrylamide) Hydrogel

How to refold inclusion bodies into natural conformation with high yield at high protein concentration is becoming one of the industrial bottlenecks in bioengineering field. In order to solve this problem, an efficient and convenient low-cost method was explored here. The temperature-sensitive poly(N-isopropyl acrylamide) (PNIPA) hydrogel was synthesized and applied into the refolding of Hen egg white lysozyme(HEWL) and recombinant bovine prethrombin-2(pThr-2) inclusion bodies.PNIPA gel disks were prepared by solution polymerization. The low critical solution temperature(LCST) of the gel was about 33℃. The swelling and deswelling equilibrium could reach for about 24h. When the gel disks were applied for the refolding of lysozyme, PNIPA gel disks improved specific activity of lysozyme by 40 % to 7012.3 U/mg within 24h.By using paraffin oil as continuous phase and Tween80 as surfactant, the faster response PNIPA gel particles were prepared by inverse suspension polymerization, the particles could reach swelling and deswelling equilibrium within about 3min. The gel particles were characterized by SEM. The particles were 0.2-0.5mm in diameter with numerous conjoint l-2μm pores spreading all over the surface of the beads. The morphology of the particles was greatly affected by the crosslinking density of the gel. There are more connected pores spreading all over the surface of the particles with higher crosslinking density. The optimum condition for lysozyme refolding with PNIPA gel particles was 30℃, 120rpm and the concentration of PNIPA gel was 80mg/mL. Under this condition, within 3h, the gel particles could increase the refolding recovery of lysozyme from 45.03% to 76.52% and the specific activity of lysozyme was 8417.4U/mg. When reused PNIPA gel particles for 8 times, the activity recovery of lysozyme still maintain at 54.98%. The gel with higher crosslinking density and higher monomer concentration could greatly improve lysozyme refolding efficiency. The novel refolding system with PNIPA enables efficient refolding especially at high protein concentrations.Kinetic model of protein refolding in vitro was established, and the model equation was solved computationally. Then with Statistica 6.0 software, the model equation was used to simulate the experimental data which were obtained from lysozyme refolding by dilution and with the addition of PNIPA gel particles. The kinetic model could well describe the process of lysozyme refolding in vitro with andAbstractwithout PNIPA gel. When PNIPA gel was added during refolding, both the refolding kinetic constant k2 and the aggregation kinetic constant kj were decreased whereas ka/ k3 was increased, which indicated the assistance of PNIPA gel to lysozyme refolding lied in the suppression of aggregation reaction.Vector with gene of recombinant pThr-2 was successfully transformed into E.coli BL21 (DE3). The fermentation conditions such as culture medium, concentration of IPTG, inoculums volume, temperature and shaking speed were investigated in detail. Results showed that HOmg inclusion bodies of recombinant pThr-2 were overproduced in E.coli BL21 (DE3) per liter broth, which increased about 40%. The optimum condition for washing of the inclusion body was 2.5mol/L urea, 0.7% TritonX-100 and 10mmol/L EDTA.The recombinant pThr-2 was expressed as intracellular insoluble inclusion bodies and must be refolded for the activity assay. The key parameters in the refolding process, such as the concentration of GSSG, urea and L-Arg, the ratio of GSSG/GSH, and the operation temperature were investigated by the classical orthogonal design combined with regression analysis. Based on the model from the regression orthogonal experiments, the parameters were successfully determined using fast hill-climbing method. Good results were obtained when refolding buffer consisted of 0.6mmol/L GSSG, GSSG/GSH 0.9, 0.1%PEG6000, and 0.5mol/L L-Arg. After dialyzed, the refolded pThr-2 was activated by Echis Carinatus snake venom and the total activity of thrombin was about 2948U/mL at op...