[Co-expression of beta-subunit with other subunits of Qbeta replicase].
In researches involving in vitro protein synthesis and self-replication system, Qbeta replicase is one of the key enzymes, which are demanded for the high availability. Qbeta replicase is a RNA-dependent RNA polymerase of Qbeta coliphage. It consists of four subunits (alpha, beta, gamma, and delta subunit), where the beta-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (alpha), elongation factors EF-Tu (gamma) and EF-Ts (delta). To increase the production of the Qbeta replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qbeta replicase. However, the beta-subunit was almost in the precipitate fraction. Considering that the four subunits of Qbeta 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 beta-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 Qbeta replicase. pBAD33-rep was constructed by cloning the beta-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 beta-subunit was co-expressed with EF-Tu-Ts. And the replicase activity assay showed this soluble enzyme is in active form. The expression of beta-subunit was enhanced by decreasing the level of inducer IPTG in co-expression, and more soluble enzyme were obtained.
Wang
Dong
D
School of Biotechnology, The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036, China. dwang@sytu.edu.cn
D004790:Enzyme Induction; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010444:Peptide Elongation Factor Tu / Q000235:genetics; D010445:Peptide Elongation Factors / Q000235:genetics; D021122:Protein Subunits; D011777:Q beta Replicase / Q000737:chemistry / Q000235:genetics*; D012995:Solubility
D010445:Peptide Elongation Factors; D021122:Protein Subunits; C023512:elongation factor Ts; D007544:Isopropyl Thiogalactoside; D011777:Q beta Replicase; D010444:Peptide Elongation Factor Tu