Eliminating DNA without sacrificing the yield and purity of your protein
Challenges in industrial protein purification
A key step in recombinant protein production is purification of the protein, which includes removal of any nucleic acids by precipitation or treatment with nucleases. Nuclease activity is greatly affected by factors such as temperature, pH and salt concentrations. DNase I and Benzonase are the most commonly-used endonucleases, but under typical protein purification conditions, these enzymes are not very efficient. Thus there is a need for nucleases that vastly improve the purification process.
On an industrial scale, it is necessary to reduce nucleic acid content of the host cell homogenate before performing any downstream protein purification processes. Large amounts of nucleic acid can increase the viscosity of the cellular homogenate, making it hard to process. For therapeutic purposes, it’s important that any contaminating nucleic acids are completely removed to prevent immunotoxic response.
Nucleic acids can be removed either by precipitation or by treatment with nucleases. However, for therapeutic proteins, nuclease treatment is preferred. In lysates, DNA which is bound to protein is protected from nuclease digestion. Increasing salt concentration is a common method to remove DNA-protein bonds. Unfortunately, the activity of commonly used nucleases such as DNase I and Benzonase are highly sensitive to salt, and are subsequently rendered inactive.
Another problem that arises during protein purification is that the process needs to be performed at low temperatures to avoid damaging the protein. However, generic nucleases are not very active at these low temperatures.
Developing a Salt-Active Nuclease that can be inactivated
To address the problem of finding a salt-tolerant nuclease, preferably with the added benefit of being active at a low temperature, ArcticZymes’ scientists took advantage of their close collaboration with The Arctic University of Norway (UiT). At UiT, enzymes from many different marine organisms have been well-characterised to understand how they have adapted to function under extreme cold conditions.
In the Arctic, there are several species of bacteria that flourish under high salt environments. Nucleases from these bacteria were tested for their level of nuclease activity under different types of conditions, e.g. at various pH levels and with different types of buffers.
A selection of promising enzymes with the desired properties were chosen for production studies at ArcticZymes’ laboratories. To develop a marketable enzyme, the gene encoding the enzyme had to be isolated and transferred to a suitable host. The genetically-engineered host strains were then grown in laboratory-scale cultures to test for their production yield and quality of the protein produced. At this stage, several challenges included finding a suitable host strain for the maximum production of the enzyme, as well as obtaining a protein that was easy to purify and did not form aggregates.
A candidate recombinant enzyme that fulfilled the desired criteria was selected for further testing. This enzyme was termed Salt-Active Nuclease (SAN) and proved to reduce viscosity in cell lysates.
However, SAN is not easily inactivated. For some protein production or molecular analysis processes, it is essential to eliminate traces of active nucleases. A highly desirable solution would consistent of an enzyme that could be conveniently inactivated.. To that end, ArcticZymes took advantage of their experience in protein engineering to create a less-stable variant, of SAN (HL-SAN). The activity of the engineered enzyme could be reduced by using heat, or completely inactivated in the presence of reducing agents.
HL-SAN is easily inactivated by treatment with a reducing agent, and its high isoelectric point (pI = 9.6), enabling easy separation of HL-SAN from a vast majority of protein targets. The optimal activity at high salinity, and the resistance to non-ionic detergents, enables HL-SAN to be used at the kinds of conditions that can facilitate dissociation of DNA from DNA-protein complexes and makes it more accessible for degradation. These features make HL-SAN the superior choice for DNA digestion in protein purification workflow, not only for laboratory sample preparation, but also for industrial bioprocessing.
ArcticZymes’ unique combination of genetic engineering capabilities and sophisticated recombinant production skills has made it possible to improve a technology for industrial scale protein production.