2. Lin J et al . (2007) Plasma cysteinylglycine levels and breast cancer risk in women. Cancer Res 67 : 11123-11127. 3. Lin J et al . (2010) Plasma homocysteine and cysteine and risk of breast cancer in women. Cancer Res 70 : 2397-2405. P2.49 Novel single-stranded DNA-binding protein from psychrophilic bacterium Photobacterium profundum SS9 Marta Nowak, Marcin Olszewski, Józef Kur Gdańsk University of Technology, Department of Microbiology, Gdańsk, Poland e-mail: Marta Nowak <[email protected]> We report the identification and characterization of the single-stranded DNA binding protein (SSB) from psychro- philic high pressure adapted bacteria Photobacterium profun- dum SS9 (PprSSB). The ssb genes obtained by PCR were cloned and PprSSB was overexpressed in Escherichia coli strain. The gene consists of an open reading frame of 552 nucleotides encoding SSB protein of 183 amino acids with a calculated molecular mass of 20.4 kDa. The amino-acid sequence of PprSSB exhibits 70% identity and 75% simi- larity to Escherichia coli SSB. In analysis by gel filtration chro- matography we show that PprSSB is functional as homooc- tamer, a structure as yet unlisted in published SSB proteins. Each monomer encodes one single-stranded DNA bind- ing domains (OB-fold). In fluorescence titrations with poly(dT), it binds single-stranded DNA with a binding site size of about 19–26 nt depending on the salt concentra- tion, and fluorescence is quenched by about 90%. When analysed by differential scanning microcalorimetry (DSC) the melting temperature ( T m ) was 60°C for PprSSB. The rare, relatively high thermostability of this cold-adapted protein and the homooctameric structure, which is unique among all known pro- and eukariotic single-stranded DNA binding proteins, could prove to be useful in various mo- lecular biology techniques.
54 Session 2. Genomics, Proteomics, Metabolomics — Medical and Environmental Implications 47 th Congress of the Polish Biochemical Society, 2012 P2.50 Effect of psychrofilic and thermophilic SSB proteins on amplification in vitro Marcin Olszewski 1 , Natalia Maciejewska 1,2 , Marta Nowak 1 , Ewelina Krajewska 1 , Sandra Zakrzewska 1 , Józef Kur 1 1 Department of Microbiology, Gdańsk University of Technology, Gdańsk, Poland; 2 Department of Medicinal Chemistry, University of Gdańsk, Gdańsk, Poland e-mail: Marcin Olszewski <[email protected]> Single stranded DNA-binding proteins (SSBs) bind coop- eratively to ssDNA, keeping them in the unwinding form, which is available for proteins involved in the processes of replication, recombination and DNA repair. SSB prevent ssDNA from forming secondary structures and from deg- radation by nucleases. There are an increasing number of studies which report the usefulness of SSBs for amplification in vitro, especially the polymerase chain reaction (PCR). A number of modi- fications to the basic PCR format have been developed in an attempt to increase amplification efficiency and specific- ity. It was shown that the use of a native ssDNA-binding protein, gene 32 protein from bacteriophage T4 or SSB from Escherichia coli
- Fall '13
- Omair Gul
- Mass Spectrometry, environmental implications, Medical and Environmental Implications, Societies Joint Meeting, Polish-German Biochemical Societies