Effect of the SA content of a novel thermo‐sensitive P(NIPAM‐co‐SA) copolymer on denatured lysozyme refolding in vitro
Effect of the SA content of a novel thermo‐sensitive P(NIPAM‐co‐SA) copolymer on denatured lysozyme refolding in vitro
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DOI：
10.1002/app.33973
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发表时间：
2011-09
影响因子：
3
通讯作者：
Jia-Yu Jin;Y. Guan;Shanjing Yao
中科院分区：
文献类型：
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作者：
Jia-Yu Jin;Y. Guan;Shanjing Yao
为改善聚N-异丙基丙烯酰胺(PNIPAM)的相转变性能，采用反相悬浮聚合法合成了P(NIPAM-co-SA)共聚物水凝胶。研究了凝胶粒子的形态、尺寸分布和热敏特性，发现SA共聚单体的加入使凝胶粒子的最大溶胀度和最低临界溶解温度(LCST)明显提高。当蛋白质浓度为2 5 0μg/mL时，P(NIPAM-co-SA)水凝胶与变性溶菌酶的最佳复性条件为：反应温度35℃，尿素浓度2M，其中含4%SA的水凝胶与溶菌酶的质量比为10：1。在此条件下，P(NIPAM-co-SA)凝胶颗粒辅助下的溶菌酶活力回收率由单纯稀释的55.6%提高到76.5%。复性完成后，凝胶颗粒易于去除和回收，重复使用5批后，溶菌酶的活力回收率仍高达61.5%。通过加入不同量的SA共聚单体，可以改变共聚物的疏水性。与PNIPAM聚合物相比，共聚水凝胶在溶菌酶复性过程中通过与蛋白质分子之间适度的疏水相互作用，更有效地抑制了蛋白质分子的聚集。以上结果表明，P(NIPAM-co-SA)是一种成本低、疏水性可调的添加剂，可用于体外表达的包涵体重组蛋白的复性。©2011 Wiley期刊，Inc.J Appl Polym Sci，2011
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. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011