Freezing-out Proteins

by Harald Zähringer, Labtimes 04/2012

Concentrated protein solutions may be separated from ice crystals via centrifugation.

A new dialysis-freezing-centrifugation procedure offers an interesting alternative to other protein concentration techniques.

Concentration of dilute protein solutions is daily routine in many laboratories. While academic researchers usually apply small-scale concentration methods, such as lyophilisation, chemical precipitation and ultracentrifugation, biotech companies additionally rely on evaporation, membrane filtration and cryo-concentration for large-scale protein concentration. With a new technique, however, cryo-concentration of proteins can also be adopted to the smaller scales in life science labs.

The basic concept of protein cryo-concentration is fairly simple, although the physics of the underlying freeze- and thaw-processes is extremely complex. The formation of ice crystals during freezing of aqueous protein solutions leads to an increase in protein concentration, which in turn gradually lowers the freezing point of the concentrated solution.

The opposite happens during thawing, when the concentrated protein solution melts before the ice crystals. Hence, all you have to do in order to concentrate proteins via cryo-concentration, is to separate the concentrated protein solution from the ice crystals during thawing − which actually sounds easier in theory than it is in practice.

Simple cryo-concentration device

A Mexican group recently came up with a simple and effective cryo-concentration system that elutes the concentrated protein solution from the frozen ice matrix via centrifugation (Virgen-Ortiz et al., Analytical Biochemistry 426, 4-12). The Mexican cryo-concentration device consists of a 20 ml plastic tube, closed with a watertight cap that fits into a slightly longer polypropylene centrifuge tube. The tube has a one millimetre edge at its upper end to fix it with the centrifugation tube. The protein solution is loaded into the tube via a small aperture in the conical bottom (the tube is turned upside down and stands on its cap for filling). The tube containing the protein solution is then stored in the freezer at -10 °C for eight hours and, subsequently, placed into the centrifuge tube and centrifuged at 4 °C and 6,150 g for 20 minutes.

The method may be further optimised by previous, rapid (two hours) dialysis of the protein solution against distilled water. The Mexican team applied the new dialysis-freezing-centrifugation technique to concentrate-diluted solutions of freshly prepared milk whey, commercial pectinase and recombinant fructan:fructan 1-fructosyltransferase (1-FFT) and attained a 9, 11 and 17-fold concentration for 1-FFT, whey and pectinase, respectively. According to Virgin-Ortiz et al., the activity of 1-FFT was not affected, suggesting that proteins are not damaged during the freeze-thawing process.

Fast procedure

And there is another point that makes dialysis-freezing-centrifugation an interesting alternative to other concentration techniques, such as ultracentrifugation: the hands-on time is just about two hours, which is pretty fast compared to approximately six hours that usually have to be scheduled for ultracentrifugation.

Last Changed: 10.11.2012