融合蛋白ELP30-Trx的表达及PEG8000对其相变温度的影响

发布时间：2018-05-02 00:52

  本文选题：类弹性蛋白 + 纯化标签　； 参考：《生物技术》2013年05期

【摘要】：目的:研究不同浓度的PEG8000对融合蛋白ELP30-Trx相变温度的影响。方法:将硫氧还蛋白(Thioredoxin,Trx)基因克隆到pET28-ELP30-Trx表达载体中,经IPTG诱导在宿主菌E.coli BLR(DE3)中表达ELP30-Trx,通过可逆相变循环(Inverse transition cycling,ITC)纯化融合蛋白。测量相变温度(T t),并检测不同浓度的PEG8000对ELP30-Trx T t的影响。结果:经酶切及测序鉴定证实成功构建了ELP30-Trx表达载体,经Westen-Blot检测表明成功表达ELP30-Trx融合蛋白。经ITC纯化后的ELP30-Trx纯度可达95%以上。5%、10%、15%、20%的PEG8000(W/V)使终浓度为25μmol/L ELP30-Trx的T t从37.9℃分别降低至34.6℃、28.5℃、14.0℃、6.0℃。结论:成功构建、表达融合蛋白ELP30-Trx,PEG8000能显著降低ELP30-Trx相变温度。通过对类弹性蛋白的研究,为类弹性蛋白的扩大应用提供理论依据。
[Abstract]:Aim: to study the effect of different concentrations of PEG8000 on phase transition temperature of ELP30-Trx fusion protein. Methods: thioredoxin Trx) gene was cloned into pET28-ELP30-Trx expression vector. ELP30-Trx was expressed in host strain E.coli BLRDE3 by IPTG. The fusion protein was purified by reversible phase change cycle reverse transition cycling. The phase transition temperature was measured and the effect of different concentration of PEG8000 on ELP30-Trx T t was measured. Results: ELP30-Trx expression vector was successfully constructed by restriction endonuclease digestion and sequencing, and ELP30-Trx fusion protein was successfully expressed by Westen-Blot. The purity of ELP30-Trx purified by ITC can reach more than 95% and 20% PEG8000W / V). The final concentration of T t of 25 渭 mol/L ELP30-Trx is reduced from 37.9 鈩，

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