Calmodulin from Homo sapiens is an a-helical calcium-binding protein that expresses to high levels in E. coli.  When the N-terminus of a calmodulin variant (N.Cam-Y) is bound to Ca2+, it undergoes a conformational change, exposing hydrophobic pockets.  This can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity.  Washing with EDTA chelates the Ca2+ ions from the protein, inducing a conformational change back to its more folded state and eluting it from the column.  In our previous work, the WW domain of human peptidyl-prolyl cis-trans isomerase (hPin1) was expressed as a fusion system C-terminal to N-Cam.Y, with a short linker between the two for proteolytic cleavage by the mutant NIa-Pro tobacco etch virus protease.  The expressed fusion protein was purified using phenyl sepharose and was then digested by NIa, which cleaves effectively to ~95% completion in most non-SDS buffers overnight.  Further purification involved either HPLC or additional phenyl sepharose column chromatography.  The result was a large amount of pure, soluble WW protein.  This fusion system proved to be extremely useful for the expression and purification of native and designed WW domain proteins.  We are currently modifying and assessing the usefulness of this procedure to express and purify zinc finger proteins, small proteins that bind to specific three-base regions of DNA.  The resultant purified zinc-finger proteins will be analyzed side by side with C-terminal histidine-tagged zinc finger proteins (purified using IMAC) for their expression levels, ease of purification and activity, the latter following the fluorescence change upon binding to the DNA of interest.  We hope that use of our novel fusion system will not alter the binding of the zinc-finger proteins, and will allow for higher expression levels with an easier purification scheme compared to the histidine tag protocol.

Development and analysis of a novel protein fusion system for expression and purification of proteins

247th National Meeting (2014)

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