Functional Qβ replicase genetically fusing essential subunits EF-Ts and EF-Tu with β-subunit

Qβ replicase, an RNA-dependent RNA polymerase of RNA coliphage Qβ, is a heterotetramer composed of a phage-encoded β-subunit and three host-encoded proteins: the ribosomal protein S1 (α-subunit), EF-Tu, and EF-Ts. Several purification methods for Qβ replicase were described previously. However, in our efforts to improve the production of Qβ replicase, a substantial amount of the β-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qβ replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the β-subunit, and in the other kind, the β-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain α-less Qβ replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme.