1. Affinity Ligands for Plasmid DNA Purification YING HAN, GARETH M. FORDE Chemical Engineering Department, Monash University, Vic 3800, Australia Materials and Methods Plasmids used in these studies are based on the plasmid pUC19 (2686 bp). This plasmid was chosen as it has a high copy number in DH5α E.coli. A lacI based ligand will be covalently immobilized via a cysteine group to a Biacore chip in order to perform initial affinity studies (dissociation constant, adsorption and desorption kinetics). Using binding data and structural biology information, optimization of the ligand will be performed. Hyperthermophiles will also be considered as possible models for biomimetic affinity ligands. Introduction The threat of an influenza pandemic has led to the need for new vaccine technologies such as plasmid DNA (pDNA) to enable faster vaccine production (1 month) than current systems (up to 9 months). The increasing demand for extremely pure, supercoiled pDNA for clinical applications creates a demand for fast, robust and reproducible purification methods. Most approaches for purifying pDNA from bacterial production systems exploit the basic physicochemical properties of supercoiled DNA (i.e. electrostatic charge) with little specificity. The specificity of affinity chromatography will overcome this drawback. Affinity purification enables the highly selective capture of target molecules in a single step hence increasing yields and reducing processing times. However, the scale-up and commercialisation of affinity chromatography requires designing ligands that meet industrial demands on robustness and regeneration. Ideally, an affinity ligand should bind pDNA reversibly with the required level of selectivity, be easy to produce, able to be immobilized onto a solid phase support and be able to withstand repeated column sanitation protocols. Biomimetics is the harnessing of ideas from nature by implementing them in another technology. In this project, naturally occurring protein-DNA interactions will be used to design biomimetic ligands for the affinity purification of DNA. dsDNA fragment loadingWash DesorptionEquilibration Fig. 2: A Biacore X sensorgram showing time (secs) along the x-axis and response units (RU) along the y-axis for dsDNA fragments binding to a lacI peptide. The rise in RU during DNA loading correlates to binding of dsDNA (20 μ g/ml, 25 μl ) to the immobilised lacI peptides. A net increase in RU can be seen which indicates binding of DNA to the ligand. The gradual decrease in RU is due to dsDNA desorption. The sudden changes in RU at [A] and [B] are due to the different compositions of the equilibration and loading buffer. The Objectives The creation of an affinity ligand, by utilizing protein-DNA interactions, for the production of extemely pure pDNA. The two ligands to be developed are: 1. A ligand with an optimized dissociation rate constant (K D = 10 -6 - 10 -8 M -1 ) for the single stage chromatographic purification of pDNA from a complex biological feedstock. 2. A tightly binding ligand for controlled release of plasmid DNA (K D < 10 -8 M -1 ). Fig. 3: A ribbon computer graphic representation of the three helix bundle of lacI protein (helix-turn- helix-loop-helix). Pure Plasmid DNA Retain beads DesorptionAdsorption Plasmid & impurities Bead Spacer Ligand impurities Wash & retain beads Plasmid target Fig.1: A schematic of affinity purification of plasmid DNA molecules. Adsorption and washing is performed under moderately acidic conditions and high salt concentration. Desorption is achieved by shifting to alkaline buffer and/or the use of eluting agents. AB