摘要
探索一种综合聚唾液酸(PSA)和聚乙二醇(PEG)优势的蛋白修饰方法。对纯化的聚唾液酸进行两步活化,先在非还原端氧化产生活性醛基,再加入胱胺形成活性巯基;活化的聚唾液酸(相对分子质量为3.4×104)和异基双功能的PEG(相对分子质量为3.5×103)形成嵌段聚合物,然后于4℃修饰尿酸酶。利用凝胶层析(Toyopearl HW-55F)对修饰后的尿酸酶进行纯化,收集相应峰进行化学法和SDS-PAGE电泳鉴定,经多角度激光光散射凝胶系统测定缀合物相对分子质量为5.214×105;相对于原始酶,修饰酶酶活保留率72.4%,体外热失活半衰期由115.5 h提高到231 h,对高温、酸碱、胰蛋白酶的耐受稳定性显著提高。
A novel protein modification method was developed by integrating the advantages of polysialic acid（PSA） and polyethylene glycol（PEG）.PSA was activated by two steps： the aldehyde group was generated by periodate oxidation at the non-reducing end,and then the active thiol group was formed using cystamine.The activated PSA（3.4 × 104） reacted with hetero-bifunctional PEG（3.5 × 103） to form a block co-polymer,and modified uricase at 4 ℃.The conjugate was purified with gel permeation chromatography and the target peak was collected and identified with chemical method and SDS-PAGE.The molecular weight of the conjugate was 5.214 × 105 through multi-angle laser light scattering gel system.Compared with native enzyme,residual enzyme activity was 72.4%,and heat inactivation half life in vitro was improved from 115.5h up to 231 h.The tolerance stability from heat,acid and alkaline,trypsin treatment was significantly improved.
出处
《生物加工过程》
CAS
2015年第2期86-92,共7页
Chinese Journal of Bioprocess Engineering
基金
江苏省自然科学基金(BK2011158)
关键词
聚乙二醇
聚唾液酸
嵌段聚合
尿酸酶
缓释
polyethyleneglycol
polysialic acid
block polymerization
uricase
controlled-release
