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The harnessing of peptide–monolith constructs for single step plasmid DNA purification

Paper ID Volume ID Publish Year Pages File Format Full-Text
35025 45069 2010 7 PDF Available
Title
The harnessing of peptide–monolith constructs for single step plasmid DNA purification
Abstract

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10−8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide–monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Keywords
Affinity ligands; Chromatography; DNA purification; Monolith; Plasmid DNA; Process scale up; Protein–DNA binding
First Page Preview
The harnessing of peptide–monolith constructs for single step plasmid DNA purification
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 2, February 2010, Pages 203–209
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering