Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Abstract

in Wiley InterScience (www.interscience.wiley.com).Peptides constructed from a-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to ''biomimics'' of the DNA binding domain of LacI. Equilibrium dissociation constants (K D ), association (k a ), and dissociation rates (k d ) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 6 1.3 3 10 210 M, 7.2 6 0.6 3 10 210 M, 4.5 6 0.5 3 10 28 M, and 6.2 6 0.9 3 10 26 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operonrepressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

“…A detailed report on the chemistry involved in production of monolith and its suitablility for pDNA purification is given elsewhere [30]. In brief, we synthesized a novel poly(GMA-co-EDMA) monolith by first mixing EDMA as the crosslinker with GMA as the functional monomer.…”

“…The following procedure was used for covalent immobilisation of the lacI peptide to epoxy group of methacrylate monolith [30]. The monolith was washed with 0.5 M Na 2 CO 3 , 1.0 M NaCl, and pH 11.5.…”

“…To achieve these conditions, a 16mer peptide representing helixII of DNA binding domain of lac repressor has been selected to use as a ligand in this study based on its binding kinetics to pDNA [30]. A reliable affinity purification requires not only optimized ligands but also a suitable stationary matrix.…”

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

“…The principle and application of the system employing the method of SPR detection is well described [49]. Coupling of the 16mer peptide to the sensor chip C1 utilised the N-terminal cysteine and the ligand thiol method as described [48,50]. …”

“…Surface plasmid resonance (SPR) assays were performed as previously described [48]. Biacore X and C1 sensor chip were both supplied by GE Healthcare (Biacore; Uppsala, Sweden).…”