Qβ RNA复制酶中亚基的共表达对β亚基溶解性的影响

Co-expression of β-subunit with Other Subunits of Qβ Replicase

在体外RNA和蛋白质合成及自复制系统的研究中 ,QβRNA复制酶作为以RNA为模板的RNA聚合酶 ,是比较重要的应用酶种之一。该酶由 4个亚基组成 ,其中只有 β亚基是由病毒基因编码 ,而其他 3个亚基都是宿主蛋白。利用普通表达载体合成Qβ复制酶时 ,得到的 β亚基几乎都是不溶性蛋白 ,从而影响了Qβ复制酶的活性和产率。为尝试提高 β亚基的溶解性 ,构建含有β亚基基因的表达质粒pBAD 33 rep ,同时利用pET2 1a(+)为表达载体表达其他 3个亚基进行共表达研究。不同亚基组合的共表达结果通过SDS PAGE分析表明 ,当 β亚基与EF Tu Ts亚基共表达时 ,溶解度有一定的提高 ,而且可溶性部分也具有复制酶活性。通过调节共表达诱导物浓度 ,相对增强可溶性 β亚基的表达 。

In researches involving in vitro protein synthesis and self-replication system, Qβ replicase is one of the key enzymes, which are demanded for the high availability. Qβ replicase is a RNA-dependent RNA polymerase of Qβ coliphage. It consists of four subunits (α, β, γ, and δ subunit), where the β-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (α), elongation factors EF-Tu (γ) and EF-Ts (δ). To increase the production of the Qβ replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qβ replicase. However, the β-subunit was almost in the precipitate fraction. Considering that the four subunits of Qβ replicase holoenzyme are in equivalent molar ratio and the amount of the subunits, ribosomal S1 and EF-Ts, being produced by the host cells is relatively low, co-expression of β-subunit with the other three subunits was performed to know whether the availability of the host subunits is the contributing factor for the solubility of the Qβ replicase. pBAD33-rep was constructed by cloning the β-subunit gene into pBAD 33, a pACYC derivative, and pET21a(+) was employed as expression vector for the three other subunits. Among the different combinations of co-expression experiments, solubility was found to slightly increase by SDS-PAGE analysis when the β-subunit was co-expressed with EF-Tu-Ts. And the replicase activity assay showed this soluble enzyme is in active form. The expression of β-subunit was enhanced by decreasing the level of inducer IPTG in co-expression, and more soluble enzyme were obtained.