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JF Hess, PG FitzGerald; Harnessing the Chaperone Activity of Alpha Crystallin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2457.
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Purpose: The in vitro chaperone activity of alpha crystallin has been demonstrated on many different lens and non-lens protein substrates. We designed experiments to utilize the chaperone activity in an effort to refold and maintain the solubility of purified recombinant proteins. Methods: Bovine alpha crystallin was purified from lens homogenates by gel filtration chromatography. Experimental proteins were expressed using bacterial pET expression vectors and purified as inclusion bodies from bacterial homogenates. Inclusion bodies were solubilized in urea containing buffers and proteins purified in the denatured state by gel filtration or ion exchange chromatography (or both). Purified experimental proteins and alpha crystalline were mixed in urea and dialyzed against simple Tris, NaCl buffers. Solubility of experimental proteins was assayed by precipitation and SDS-PAGE. Results: Inclusion of alpha crystallin, at various concentrations, to purified proteins in urea followed by dialysis to remove the urea was found to be an effective method to maintain the solubility of purified proteins. We have assayed several different proteins and when purified, each has been found to precipitate following removal of urea. However, addition of alpha crystalline to the protein prior to dialysis prevents the precipitation of the purified protein. Conclusion: The chaperone activity of alpha crystallin is able to survive treatment with high concentrations of urea and in vitro will prevent the precipitation of purified proteins that commonly occurs during dialysis to remove urea. Thus, alpha crystallin may be used in vitro to both maintain solubility and aid in the proper folding of recombinant proteins.
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