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- W2389602178 abstract "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." @default.
- W2389602178 created "2016-06-24" @default.
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- W2389602178 date "2004-01-01" @default.
- W2389602178 modified "2023-09-24" @default.
- W2389602178 title "Co-expression of β-subunit with Other Subunits of Qβ Replicase" @default.
- W2389602178 hasPublicationYear "2004" @default.
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