一种新的大肠埃希菌蛋白质快速合成方法

目的 在大肠埃希菌中建立同时进行PCR克隆和重组蛋白质快速表达的方法。方法 通过TA载体将包含目的基因和全部蛋白表达元件的蛋白表达单位引入大肠埃希菌后，无需使用任何限制性酶，直接在大肠埃希菌中表达编码的蛋白质。结果 通过小规模或大规模表达多种不同蛋白质，证实了该方法的有效性和可靠性。使用标准光谱（280 nm）定量方法分析纯化的蛋白质，发现绿色荧光蛋白的可溶性蛋白表达量达到30 mg/L，血管内皮生长因子特异性 VH的可溶性蛋白表达量达到20 mg/L。结论 该方法设计灵活，且易于进行多种蛋白质的并行表达，为快速筛选理想的蛋白突变体提供了一新途径。

A novel procedure for rapid protein synthesis in E. coli

Objective  To establish a novel method of simultaneous PCR cloning and rapid recombinant protein expression in E. coli. Methods  Protein Expression Unit (PEU) containing target gene and all essential elements for protein synthesis was introduced into E. coli through TA-vector. Without restriction enzymes, protein encoded by PCR was directly synthesized in E. coli. Results  The effectiveness and reliability of this method were demonstrated by successful expression of many different proteins at either small or large scales. Quantified by the standard spectra (280 nm) analysis of purified proteins, soluble protein level reached 30 mg/L for green fluorescence protein and 20 mg/L for vascular endothelial growth factor-specific VH. Conclusions  The established approach is flexible by design and easy-to-perform for producing multiple proteins in parallel, providing a rapid screening for desirable protein variants.