Directed evolution of novel polymerase activities: mutation of a DNA polymerase into an efficient RNA polymerase.
The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates.
Xia
Gang
G
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Chen
Liangjing
L
Sera
Takashi
T
Fa
Ming
M
Schultz
Peter G
PG
Romesberg
Floyd E
FE
D000595:Amino Acid Sequence; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology; D014760:Viral Fusion Proteins / Q000235:genetics
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D019914:Taq Polymerase; D012321:DNA-Directed RNA Polymerases