(510o) Binding Mechanism of Affinity Ligands for Purification of Plasmid DNA | AIChE

(510o) Binding Mechanism of Affinity Ligands for Purification of Plasmid DNA

Authors 

Han, Y. - Presenter, Monash University
Forde, G. M. - Presenter, Monash University


The demand for plasmid DNA (pDNA) has increased dramatically in response to rapid advances in the use of pDNA in gene therapy and DNA vaccination. Gene therapy, a therapeutic method in which nucleic acids are introduced into human cells to cure specific diseases, promises to revolutionize the treatment of inherited and acquired diseases. In genetic engineering, a vector is a virus or plasmid that carries a molecule of recombinant DNA into a cell, thus transforming it. The virus is classified as the viral vector, and the plasmid is referred to as the non-viral vector. Both viral vectors and non-viral vectors have been developed to deliver nucleic acids to target cells. However, the use of viral vectors has raised safety and regulatory concerns over their toxicity and immunogenicity after some deaths were reported. This has led to the study of plasmid DNA (pDNA) as a non-viral vector which has the advantages of being free from specific safety concerns associated with viruses and of being simpler to develop. The number of approved gene therapy protocols using plasmid DNA has almost doubled since 2000. In 2000, 8.4 % of gene therapy trials worldwide employed DNA (plasmid or naked DNA). In contrast, in 2004, 14.4 % of gene therapy trials employed pDNA or naked DNA as the gene transfer vector. The worldwide gene therapy trials have resulted in the demand for large-scale production of purified gene vectors.

Moreover, the potential use of pDNA in vaccines has been shown through the expression of specific antigens on cell membranes that help to stimulate the immune system's response and memory. The first reported use of DNA outside of clinical trials was as recent as 2003, where pDNA was used to immunize California Condors against the West Nile virus. Zoo veterinarians report absolutely zero negative effects and preliminary blood tests suggested that the birds exhibit a ?fantastic immune response'. As a result, there has been an increasing demand on the biotechnology industry to supply purified pDNA for vaccine and gene therapy.

In addition, according to the regulatory agencies' guidelines, such as those set forth by the Food and Drug Administration (FDA) in the USA, the plasmid DNA for gene therapy must be available as a highly purified product to minimize any side effects on humans. The FDA specifies four classes of molecules that must be qualified and minimized during plasmid purification from E coli cells: genomic DNA, RNA, protein, and endotoxin. To meet the strict regulations and specifications of plasmid DNA, there is a need for highly selective processes to purify clinical grade pDNA.

In general, the process for producing pDNA follows the three phases of fermentation, cell disintegration and purification. Plasmid DNA is usually produced in E coli cells by fermentation process, followed by cell disintegration using alkaline lysis methods because of the shear force sensitivity of polynucleotides and expensive enzymatic disintegration. After the first two processes, the concentration of pDNA is not more than 3% of the clarified cell lysate even with high copy number plasmid. As a result, a further efficient purification process is essential to quickly capture, concentrate and purify the pDNA.

Affinity-based purification of plasmid DNA owns unique high resolution to be considered to better purify pDNA. Affinity chromatography is theoretically capable to selectively purify biomolecules in a single process. Its mechanism involves stationary immobilized ligands on a solid matrix. A mobile phase containing target molecule is flowed through under conditions that favour its specific binding to the immobilized ligand. Unbound and weakly bound substances are washed off and the target substances can be recovered by changing the conditions to those which favour its adsorption. So affinity chromatography could eliminate steps, increase yields and downsize capital and improve process economics.

However, without suitable ligands, no affinity purification protocol can be successful. Therefore, such protocols require the design of ligands with optimised affinity to the target product pDNA. The design of highly selective and stable synthetic affinity ligands will have a significant impact on future applications of affinity chromatography. Obtaining the smart ligands will enable pDNA purification systems to be simplified by combining purification, concentration and clarification into a single process.

To find out smart ligands, the formation of triple helices between oligonucleotides linked to a chromatographic matrix and duplex sequences present on the plasmid molecule has been explored for affinity chromatography, but only at laboratory and the formation of the triplex is slow, so the process is time consuming. Initially, proteins from two of the eight structural groups, the zinc-co-ordinating group (zinc finger protein) and helix-turn-helix group (lacI repressor protein) seem to be the most suitable for pDNA binding protein. Zinc finger proteins have previously been reported to sequence-specifically bind and immobilise pDNA, the subsequent recovery of the pDNA has proved problematic.

Besides these methods, based on the knowledge of the structure and biological specificity, a naturally occurring LacO-LacI binding could be used for purification of plasmid DNA. A lac repressor (lacI) displays affinity for lac operator (lacO) sequences. This binding occurs naturally when repression of the lac operon is achieved by halting the binding of RNA polymerase in a pretranscriptional complex with the promoter by the lacI being bound to the first lacO. In the present system, the lacI peptide exhibits a specific affinity for lacO regions contained in a pUC19 plasmid.

In this work, the design of lacI based peptide as affinity ligands for purification of plasmid DNA were presented. Besides, this paper also demonstrates the novel cost-effective approach ? surface Plasmon resonance based Biacore technology which has been employed in studying in detail the kinetics of the binding between plasmid DNA and peptide ligands. In order to investigate the kinetics of the binding between pDNA and a ligand, the surface plasmon resonance (SPR) biosensor technique was employed. SPR is a phenomenon that occurs when light is reflected off thin metal films. A fraction of the light energy incident at a sharply defined angle can interact with the de-localized electrons in the metal film (plasmon) thus reducing the reflected light intensity. The refractive index of the SPR-active gold coated glass slides changes very sensitively in the presence of biomolecules on the gold surface, and the changes induce a shift in the resonance angle commonly called ?the surface plasmon resonance angle?. The interactions between biomolecules immobilized on the sensor surface (peptide here) and biomolecules in an aqueous buffer flowing over the surface can be quantified by measuring the angle drift. This technique offers significant advantages such as tiny amount of sample required, being label-free and real-time for characterizing biomolecular interactions.

In addition, the characterization and mechanism of the binding between pDNA and peptide were analysed and discussed in this study. The protein affinity approach for pDNA purification is extended to utilize the interaction between the lac operon sequence contained in the pDNA and its repressor, the lacI protein. The ligand designed with an optimised dissociation constant could facilitate the successful operation of affinity chromatography for purification of pDNA. The optimal dissociation constant for an affinity binding mechanism for use in a chromatographic system is 10?6 M?1 - 10?8 M. If the dissociation constant is greater than this value, non-target molecules can be co-purified as the mechanism is not selective enough for the target. If it is too low, production yields are low. The ligands designed will be specific enough so that the target pDNA is selectively bound without the co-purification of other ingredients (gDNA, RNA, protein, and endotoxins) with higher yield. Based on the kinetic results from SPR based Biacore system, affinity driven ligand selection was also discussed here.

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