Unlike the more
familiar chromosomes, which are long strands of DNA, plasmids are small
circles of DNA found in bacteria, which are separate from its main genetic
material. By adding a gene of interest to an isolated plasmid and
reinserting it back into a bacterium, molecular biologists can exploit the
ability of bacteria to replicate rapidly to make copies of the new gene.
Administering these engineered plasmids to a patient with a genetic disorder
could replace a missing or damaged gene with a working copy. However, as
chemical engineers know only too well, purity is a limiting factor in many
processes.There are several methods for cleaning up plasmids for gene therapy applications. However, these tend to be labour-intensive require several demanding steps to avoid leaving potential contaminants, such as toxins from the host bacteria. They also yield only relatively small amounts of plasmid material. "Plasmid DNA is a potentially attractive route for delivering genes," explains Professor Nigel Slater, who is working with Dr Sid Ghose on the Cambridge side of the collaboration. "But manufacturing would benefit from a simple process that yields a very pure preparation of the plasmid DNA. The bacterial cell contains many contaminants, such as toxic lipopolysaccharide (LPS), toxins from the cell wall."
Slater and his colleagues at Cambridge are working with molecular biologist Dr Anna Hine and Dr Richard Darby at Aston University to find a simple way to obtain pure plasmid DNA rapidly and with few process steps. The key to their new approach is to exploit proteins which bind exclusively to specific target regions, or 'sequences', within the plasmid DNA. These protein groups are attached at one end to a solid support material, such as beads of the polymer gel agarose, then as the bacterial cell mixture passes over them the other end of the protein unit latches on to the plasmid DNA only, ensnaring it while other compounds, including the toxins are simply washed off. The plasmid DNA can then be released in pure form from the beads.
Slater and Hine have worked together using two approaches to this problem. In the first, they constructed a protein in which a finger-like protein unit, known as a zinc-finger transcription factor is coupled to the enzyme GST (glutathione-S-transferase). The zinc-finger latches on to the target recognition DNA sequence in the modified plasmid DNA. The researchers can then capture this protein-DNA complex on an agarose bead carrying glutathione groups. In the second, simpler approach, the researchers replaced the zinc-finger with another protein known as LacI. LacI binds strongly to a target DNA sequence, known as the <I>lac</I> operator. As they explain, the first approach was a good model system to establish the feasibility of the programme and subsequently, to establish technical parameters, but the protein binds so tightly to the DNA, that it may be difficult to remove at the end of the process. The second approach is far more suitable for actual plasmid purification because the strong binding is entirely reversible and after purification, the DNA can be released from the protein at will using a solution of the sugar molecule allolactose , or one of its synthetic analogues. These compounds change the shape of the LacI protein, which shakes off the pure plasmid, while the LacI protein is left behind, still bound to the solid support.
Hine's team addressed the molecular biology aspects of the project on a small scale and Slater's group tackled the engineering aspects of the project, such as how to scale the work up for commercial viability. They have found that although they can successfully extract plasmids with the zinc finger approach, recovery of the purified material is relatively difficult. However, having d emonstrated proof of principle, they turned to the LacI method and have now developed several techniques for producing highly pure DNA in a simple affinity process. The LacI system works in a packed bed chromatography system. In this system, a glass column is packed with silica gel which absorbs different materials at different rates as a mixture is flushed through with solvent so th ey can be tapped off one after the other. This type of chromatography works on pure, clarified and filtered solutions. The results demonstrate the possibility of using the affinity system in a scalable process and allowing the plasmid DNA to be extracted in a single step ready for storage.
"This is a technique called affinity chromatography and is widely used to purify other biological substances such as antibodies," says Slater. "It is a very attractive and powerful technique but there is no equivalent process for DNA which can be generically operated at a production scale to make grams of DNA."
The team has now further developed their approach into an expanded bed absorption (EBA) system. EBA systems are commercially available and commonly used for separating out components in blood and other biological mixtures. The EBA system offers process advantage by allowing mixtures to be processed that have not been clarified so they can be loaded directly on to a chromatography column in which the adsorbent material is suspended in the flow stream. This, the researchers explain, allows the cell debris and other materials to be flushed through quickly. The EBA system therefore works more effectively than the packed bed approach allowing the team to extract plasmid DNA directly from the bacterial cells.
"From an engineering viewpoint the study has extended fundamental scale-up principles to large macromolecular products," Slater told Newsline, "bringing together the design of chromatography materials, considerations of mass transfer and fluid flow and sophisticated process analysis."
"This is the first demonstration of protein affinity purification procedures for DNA manufacture," adds Slater, "The procedures we've been working on are largely restricted to DNA but some of the problems we've faced, for example how to adsorb very large macromolecules on to beads designed for the adsorption of small proteins, are generic." He adds that there is crossover with work he and his colleagues are doing on affinity purification of viruses and synthetic nanoparticles for drug delivery."