The need for a purified protein in sub-milligram to milligram quantities arises quite frequently in biological and biomedical research. Examples include antibody production, enzyme characterization, protein-protein interaction studies, and assay development. Due to the substantial decrease in time and effort, the majority of laboratories have opted for recombinant protein expression in prokaryotic or eukaryotic systems in lieu of classical chromatographic techniques for protein purification from cells or tissue.
Of all the expression systems available, by far the simplest protocols involve bacterial expression. Although bacterial expression may not be the appropriate choice in cases where glycosylation or specific protein folding is crucial, there are many instances where it is. Some reasons for this include high recombinant protein yield, ease of use, and the sizeable choice of expression vectors available for essentially any set of circumstances. Some of the most useful types of expression vectors are those that permit the expression of a protein covalently linked to a particular moiety as a fusion protein.
The main reason for expressing a protein of interest as a fusion protein is for ease of purification, usually by using an affinity reagent that binds to the fusion partner coded for by the vector. In addition to allowing for a simple method of protein expression and purification, many fusion protein vectors are designed such that the fusion partner can be removed from the protein of interest, thus eliminating any downstream interference. This is most often accomplished by the insertion of a protease-specific cleavage site between the fusion partners. A well-used example is the pGEX-T series of vectors from Amersham Biosciences (now GE Healthcare). Fusion proteins expressed from these vectors consist of a user-selected protein attached to glutathione S-transeferase (GST) and separated by a thrombin-specific cleavage site.
Once the fusion protein is purified using a glutathione affinity matrix, the GST moiety can be cleaved off by an overnight incubation at room temperature with thrombin. The protein preparation now consists of the protein of interest, GST, uncleaved fusion protein, and thrombin. Both free GST and uncleaved fusion protein can be removed using the glutathione affinity matrix, but thrombin remains. Removal of this serine protease could be a bit of a problem if not for a very nicely designed little device called the HiTrap Benzamidine FF affinity column (Amersham Biosciences). Benzamidine is known to many researchers as an effective serine protease inhibitor. A less known fact is that the very properties that makes benzamidine a good inhibitor, i.e. tight and specific binding, also make it an excellent affinity reagent.
HiTrap Benzamidine FF affinity columns come in 1 ml and 5 ml sizes. I have used the 1 ml column and found it to be very easy to use and quite effective. A package of 2 columns includes a good variety of Luer adapters, tubing connectors, and plugs. Although the columns are sealed and can’t be opened, the affinity matrix is quite robust and can be reused many times. And, given the small amount of thrombin used in the fusion protein cleavage reaction, the capacity of the column is more than sufficient for all but the largest protein preps.
An additional use of the HiTrap Benzamidine FF columns is to remove serine proteases from serum, lysates and monoclonal antibody supernatants. This can be a very effective alternative to protease inhibitors, which may add undesirable qualities to a reagent.
Overall, the HiTrap Benzamidine FF columns are a very efficient and economical means of removing serine proteases and are particularly useful for thrombin removal following the cleavage of fusion proteins.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center