Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.
The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland. Electronic address: stefanska@aabiot.com.
Aleksandra
A
Stefanska
Collection of Plasmids and Microorganisms, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland. Electronic address: szuster@biotech.ug.gda.pl.
Anna-Karina
AK
Kaczorowska
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland. Electronic address: magdalena.plotka@biol.ug.edu.pl.
Magdalena
M
Plotka
Matis ohf, Vinlandsleid 12, Reykjavik 113, Iceland. Electronic address: olafur@matis.is.
Olafur H
OH
Fridjonsson
Matis ohf, Vinlandsleid 12, Reykjavik 113, Iceland; Faculty of Life and Environmental Sciences, University of Iceland, Sæmundargötu 2, Reykjavik 101, Iceland. Electronic address: gudmundo@matis.is.
Gudmundur O
GO
Hreggvidsson
Prokazyme ehf, Vinlandsleid 14, Reykjavik 113, Iceland. Electronic address: sigridur.hjorleifsdottir@orfgenetics.com.
Sigridur
S
Hjorleifsdottir
Prokazyme ehf, Vinlandsleid 14, Reykjavik 113, Iceland. Electronic address: jakob@arkea.is.
Jakob K
JK
Kristjansson
A&A Biotechnology, Aleja Zwyciestwa 96/98, 81-451 Gdynia, Poland. Electronic address: sd@aabiot.com.
Slawomir
S
Dabrowski
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland. Electronic address: tadeusz.kaczorowski@biol.ug.edu.pl.
Tadeusz
T
Kaczorowski
D000595:Amino Acid Sequence; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D017900:Myoviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004279:DNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D011938:Rec A Recombinases
PCR; RecA protein; Strand displacement; Thermus phage