Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.
Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta 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.
Kita
Hiroshi
H
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Cho
Junghwa
J
Matsuura
Tomoaki
T
Nakaishi
Tomoyuki
T
Taniguchi
Ichiro
I
Ichikawa
Takehiko
T
Shima
Yasuhumi
Y
Urabe
Itaru
I
Yomo
Tetsuya
T
D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D024861:Genetic Enhancement; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits; D011777:Q beta Replicase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D011777:Q beta Replicase