Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: The alteration of the product by an affinity tag

The efficiencies of different procedures for purification of the capsid pro tein (CA) of Mason-Pfizer monkey virus are compared. Plasmids encoding both wild-type CA and two C-terminally modified sequences of CA suitable for af finity chromatography purification were prepared. CA was expressed in Esche richia coli (i) as a wild-type protein, (ii) C-terminally extended with a s ix-histidine tag (CA 6His), and (iii) as a protein containing a C-terminal fusion to a viral protease cleavage site followed by a six-histidine tag (C A 6aa6His). Electron microscopy was used for comparison of the resulting pr oteins, as CA is a structural protein with no enzymatic activity. We have f ound that these C-terminal fusions dramatically influenced the properties a nd morphology of structures formed by CA protein in E. coli. The formation of amorphous aggregates of CA was abolished and CA 6His and CA 6aa6His prot eins formed organized structures. CA and CA 6aa6His accumulated in bacteria in inclusion bodies as insoluble proteins, CA 6His was found in a soluble form. Both six-histidine-tagged proteins were purified using affinity chrom atography under either native (CA 6His) or denaturing (CA 6aa6His) conditio ns. CA protein was purified under denaturing conditions using gelfiltration chromatography followed by refolding. All proteins were obtained at a puri ty > 98%. Both aforementioned C-terminal extensions led to dramatic changes in behavior of the products and they also affected the tendency to form or ganized structures within E. coli. We show here that the widely used histid ine anchor may significantly alter the properties of the protein of interes t. (C) 2001 Academic Press.