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Chromatography 2016
September 21-23, 2016
Volume 7, Issue 5(Suppl) J Chromatogr Sep Tech 2016
ISSN: 2157-7064 JCGST, an open access journal
conferenceseries .com
September 21-23, 2016 Amsterdam, Netherlands
World Congress on Chromatography
Aptamer-based affinity chromatography as a rapid, single step method for purification of native proteins
Svetlana M Krylova, J Bao, S Boloborodov, O Borisade and S N Krylov
York University, Canada
Isolation and purification of recombinant proteins is one of major tasks of modern biotechnology. Isolation of enzymes
and antibodies requires conditions that could preserve biological activities of proteins. Often fusion of proteins with His-,
GST-, and MBP-tags is used to facilitate their isolation by affinity chromatography. However, the tags, may interfere with the
application of the protein while there removal is often accompanied by protein’s loosing its biological activity. We developed
aptamer-based affinity chromatography allowing isolation of the recombinant proteins from the crude cell lysate as a quick
method yielding native biologically active enzymes. DNA aptamers to AlB protein were developed and characterized by Kinetic
Capillary Electrophoresis (KCE). Synthetic DNA aptamers with Kd values in the nanomolar range were used for selective
binding and isolation of AlkB from the cell lysate. Specifically, gold (DE3) bacterial culture of cells, expressing E. coli AlkB
protein was loaded on aptamer-modified magnetic beads (immobilized though a biotin-streptavidin link). The unwanted
components of the cell lysate were removed by washing the beads. AlkB was eluted using different solutions with high ionic
strengths. The results were compared with the activity and yield of the enzyme purified using standard tag-based methods
of protein purification. Our new method was also succesfully repeated for isolation and purification of MutS protein. In my
presentation, I will discuss the CE based aptamer development technology, and I will demonstrate the potential of using
aptamers for purification of enzymes from cell lysates in a single simple step, providing biologically active pure recombinant
proteins.
Biography
Svetlana M Krylova completed her PhD from the Russian Academy of Sciences. She has over 10 years of research leadership experience in the area of Medical
Diagnostics and Drug Development in biotechnology and pharmaceutical companies in Canada. She has been a contract faculty member at York University
in Toronto since 2008. She is leading research projects in the area of Bioanalytical Chemistry as a Senior Research Associate in the Centre for Research on
Biomolecular Interactions at York University.
Svetlana M Krylova et al., J Chromatogr Sep Tech 2016, 7:5(Suppl)