黄凯宗，李晶晶，李巍，葛慧华，王文研，张光亚

华侨大学生物工程与技术系，厦门 361021

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

黄凯宗，李晶晶，李巍，葛慧华，王文研，张光亚

华侨大学生物工程与技术系，厦门 361021

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25 µmol/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。

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

Abstract: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.

Keywords:elastin-like polypeptides, non-chromatographic purification, inverse transition cycling, transition temperature,purification tag

蛋白质分子大规模分离纯化是当前生物工程中的关键技术问题，纯化过程所需成本约占总成本的60%~70%，色谱法为目前分离蛋白较成熟的方法。融合标签技术极大地简化了重组蛋白质的纯化过程，它通过融合一段特定的多肽 (如组氨酸标签) 进行融合表达，并通过适当的亲和力或者对配体有高特异性，融合蛋白可以与固相基质上的特异配基结合，从而使重组蛋白质得以纯化[1]。但在实际应用中，亲合色谱法成本很高，不仅需要特殊设备，须对固相基质进行定制，难以放大到工业规模，且纯化效率低[2]。……