Effect of the SA content of a novel thermo-sensitive P(NIPAM-co-SA) copolymer on denatured lysozyme refolding in vitro

摘要

A novel hydrogel of P(NIPAM-co-SA) copolymer was synthesized by inverse suspension polymerization by adding sodium acrylate (SA) to improve the phase transition properties of poly(N-isopropylacrylamide) (PNIPAM). The morphologies, size distribution and thermosensitive characteristics of gel particles were studied and the maximal swelling ratio and LCST (Lower Critical Solution Temperature) of gel particles increased obviously with the addition of SA comonomer. When the protein concentration was 250 μg/mL, the optimized refolding conditions of denatured lysozyme with P(NIPAM-co-SA) hydrogel were that operating at the temperature of 35°C and a urea concentration of 2M, in which the mass ratio of P(NIPAM-co-SA) hydrogel with 4% SA copolymerized to lysozyme was 10: 1. Under the optimized conditions, the activity recovery of lysozyme increased to 76.5% assisted by P(NIPAM-co-SA) gel particles compared with 55.6% by simple dilution. When refolding finished, the gel particles could be removed and recovered easily and the activity recovery of lysozyme was still as high as 61.5% after reused for 5 batches. With the addition of different amounts of SA comonomer, the hydrophobicity of the copolymer could be varied. Then the copolymerized hydrogel inhibits protein molecules aggregation more effectively through the moderate hydrophobic interactions between copolymers and protein molecules in the course of lysozyme refolding compared with the presence of PNIPAM polymer. All results above demonstrate that the P(NIPAM-co-SA) is a cost effective additive with tunable hydrophobicity for application in the refolding of recombinant proteins expressed as inclusion bodies in vitro.