摘要
以类弹性蛋白多肽(ELPs)为标签的非色谱纯化重组蛋白显示出良好的应用前景.本文使用自行设计的新型ELPs纯化标签,以低浓度盐诱发其可逆相变,从而更温和、高效地纯化融合表达的1,3-丙二醇氧化还原酶(PDOR).在测得ELPs-PDOR融合蛋白在不同浓度Na2CO3溶液中相变温度的基础上,选取0.8 mol/L Na2CO3溶液,在室温下使ELPs发生可逆相变,与目的蛋白PDOR一同聚集,经可逆相变以离心法得以纯化,得到纯度约98%的纯酶,纯化倍数可达到8倍以上,是组氨酸标签的7倍多.融合酶的酶学性质表明ELPs作为纯化标签对目标蛋白空间结构影响微小,融合蛋白可直接作为酶催化化学反应.低盐诱发相变的方法,可避免盐浓度过高对酶结构及性能产生的不利影响,因此,在重组酶纯化上更具有优势.
Elastin-like polypeptides have been widely used as the purification tag. Non-chromatographic purification of ELPs fusion proteins, termed inverse transition cycling （ITC）, exploits the reversible soluble-insoluble phase transition behavior imparted by the ELPs tag. This study found that the novel ELPs tag could purify 1,3-propanediol oxidoreduetase （PDOR） efficiently. The ELPs aggregated with 0.8 mol/L Na2CO3 at room temperature, and then PDOR, which was linked with the ELPs, was purified by ITC. The purity could reach about 98%, and the purification fold was above 8, about 7 times that of His- tag purification method. By comparing the properties of the purified fusion ELPs-PDOR and His-PDOR, we found that ELPs tag hardly affects the target protein, suggesting that it can be used during the biocatalysis process. The method with low salt concentration to induce the phase transition of ELPs can avoid potential adverse effect on enzyme structure and property, thus being advantageous in protein purification. Fig 3, Tab 2, Ref 17
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2013年第4期709-712,共4页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(20806031)
福建省高校新世纪优秀人才支持项目(07176C02)资助~~
关键词
类弹性蛋白多肽
纯化标签
可逆相变
重组酶纯化
非色谱法
elastin-like polypeptides
purification tag
inverse temperature transition
recombinant enzyme purification
non-chromatographic
