类弹性蛋白多肽的从头设计、非色谱纯化及盐效应  

De novo design,non-chromatographic purification and salt-effect of elastin-like polypeptides

作　　者：黄凯宗[1] 李晶晶[1] 李巍[1] 葛慧华[1] 王文研[1] 张光亚[1] 

机构地区：[1]华侨大学生物工程与技术系,厦门361021

出　　处：《生物工程学报》2011年第4期653-658,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.20806031);福建省自然科学基金(No.2009J01030);华侨大学高层次人才科研启动项目(No.10BS220)资助~~

摘　　要：旨在克隆、表达与纯化类弹性蛋白多肽,并测定类弹性蛋白的相变温度对不同的盐敏感程度。从头设计了类弹性蛋白多肽的序列并人工合成其编码基因片段,克隆至改造后的表达载体pET-22b(+)中,构建重组表达载体,转化至大肠杆菌BL21(DE3)中并诱导表达,采用可逆相变循环经高速离心对其进行纯化,并考察了盐类型及浓度对类弹性蛋白相变温度的影响。结果表明:0.4 mmol/L的Na2CO3能使25μmol/L类弹性蛋白多肽[KV8F-20]相变温度降低至19℃,此类弹性蛋白多肽序列有望开发成一新型纯化标签,为今后重组蛋白的非色谱分离纯化奠定基础。Elastin-like polypeptides（ELPs） are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin.It was a promising tag for recombinant protein purification.Here,we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b（＋）.Then,we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21（DE3）.Upon induction by Isopropyl β-D-Thiogalactoside（IPTG）,ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling.The influences of salts types and concentrations on ELPs were also determined.The results showed that the transition temperature of the [KV8F-20] decreased to 19 °C by 0.4 mmol/L Na2CO3.Due to its small molecular weight and sensitivity to salt,the ELPs might be a useful purification tag,which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.

关 键 词：类弹性蛋白多肽 非色谱法纯化 可逆相变循环 相变温度 纯化标签 

分 类 号：TQ936[化学工程]