Title
Purification of supercoiled G-quadruplex pDNA for in vitrotranscription
Author
Tiago Santos, Patrícia Pereira, Fani Sousa, João A. Queiroz, Carla Cruz
Year
2016
Journal
Separation and Purification Technology
Abstract
The formation of G-quadruplex in G-rich regions of DNA can be induced by transcription and is formed within plasmid transcribed in Escherichia coli. The G-loops are more evidenced on supercoiled (sc) topology than on relaxed (oc) or linearized (ln) plasmid isoforms. Thus, the present work reports different purification strategies to efficiently purify the pPH600 sc isoform from other plasmid topologies and host contaminants present in a clarified E. coli lysate. To accomplish this purpose, two affinity supports, l-tyrosine-Sepharose and l-tryptophan-Sepharose were prepared by linking l-tyrosine andl-tryptophan onto epoxy-activated Sepharose CL-6B and were further characterized. The commercial support l-arginine Sepharose was also used to purify sc pPH600 since it has already been efficiently applied to separate sc isoforms of other plasmids using mild binding and elution conditions.
Instrument
J-810
Keywords
Circular dichroism, Biochemistry