Australian researchers have come up with a simple way to release whole proteins from ion exchange stationary phases: turn the heat down. According to the researchers, such temperature-responsive stationary phases could form the basis for an inexpensive, environmentally-friendly way to isolate and purify proteins at industrial scales.

To produce these novel stationary phases, researchers led by Brad Woonton at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia's national science agency, took advantage of poly(N-isopropylacrylamide) (poly-NIPAAm). This is a well-known temperature-responsive polymer that undergoes a phase transition at around 30°C, changing from soluble to insoluble as the temperature rises. Although poly-NIPAAm has already been used to produce numerous temperature-responsive stationary phases, it had never before formed the basis of an ion exchange resin for separating whole proteins.

Woonton and his team combined poly-NIPAAm with small amounts of tert-butylacrylamide, which enhanced the temperature-sensitivity of poly-NIPAAm, and acrylic acid, which provided cation exchange abilities. They then coated this co-polymer onto agarose beads to form the novel ion exchange stationary phase.

Ordinarily, ion exchange resins are coated onto silica beads rather than agarose, but silica beads tend to be quite expensive and not particularly robust. They are therefore mainly used for laboratory analyses. So in order to produce a more robust stationary phase for potential industrial applications, Woonton and his team turned to agarose beads.

The researchers tested this stationary phase with lactoferrin, a protein found in milk that possesses anti-microbial activity and is found in various health products. They quickly discovered that lactoferrin would bind much more readily to the stationary phase at 50°C than at 20°C (by a factor of three).

As such, most of the lactoferrin binding to the stationary phase at 50°C could be released by just reducing the temperature in the chromatography column to 20°C. Any lactoferrin still remaining on the stationary phase could then be released by simply passing a mild salt solution through the column.

Proteins bound to conventional ion exchange stationary phases are usually released by passing fairly concentrated salt solutions through the column. Indeed, Woonton and his team found that this is exactly what they needed to do if they wanted to release the lactoferrin at 50°C.

All these tests were conducted at ph 6.5, giving the lactoferrin a positive charge. When Woonton and his team mixed lactoferrin with two related proteins that are negatively charged at this pH - alpha-lactalbumin and beta-lactoglobulin - they found that only lactoferrin bound to the stationary phase at 50°C.

The results from all these tests suggest that lactoferrin binds to the stationary phase in two complementary ways. Usually, proteins bind to ion exchange stationary phases solely via electrostatic interactions between the opposite charges of the protein and the ion exchange resin. But in this case, in addition to the electrostatic interactions, lactoferrin also appears to bind via interactions between hydrophobic (water-hating) residues on its surface and hydrophobic regions on poly-NIPAAm. These hydrophobic regions only appear above 30°C, when the polymer chains curl up to form insoluble, globular particles. Below 30°C, they disappear, as the polymer chains unfurl to become extended hydrophilic (water-loving) coils.

So at 20°C, the lactoferrin only weakly binds to the stationary phase via electrostatic interactions, but at 50°C it strongly binds via both electrostatic and hydrophobic interactions. In the case of alpha-lactalbumin and beta-lactoglobulin, the hydrophobic interactions alone are not strong enough to bind them to the stationary phase. Remove one of the interactions and the proteins either don't bind at all or are easily released. 

By removing the need for concentrated salt solutions to release bound proteins, the researchers claim that their temperature-responsive stationary phase should simplify the whole process of separating cationic proteins from complex protein solutions via ion exchange chromatography. This could be especially useful for industrial-scale protein separation processes, with Woonton specifically highlighting the potential for fractionating the proteins in whey, a milky by-product of cheese manufacture.

Related links:

• Journal of Chromatography A (Article in Press): "Development of a temperature-responsive agarose-based ion-exchange chromatographic resin"   

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.