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P. Santhoshkumar, Y. Sreelakshmi, K. Sharma; Immobilized Metal Affinity Chromatography (IMAC): An in vitro technique to study lens protein–protein interactions . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3973.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Protein–protein interactions play an important role in maintaining lens transparency and in the development of cataract. The objective of this study is to develop a method to immobilize the protein complexes for easy isolation, detection and analysis of the interacting proteins. Methods: Recombinant αA– and αB crystallins were used to demonstrate the technique. To immobilize the complex, his–tag was introduced to N–terminal end of αA–crystallin (6H–αA) using PCR and the proteins were expressed in E.coli BL21(DE3)pLys cells. 6H–αA protein was purified using Nickel affinity gel (Sigma) and the wild type αB was purified by means of ion–exchange and gel filtration chromatography. αB–crystallin was labeled with a fluorescent dye Alexa Flour 568 (Molecular Probes). The structure of wild–type and his–tagged proteins were analyzed using CD spectroscopy. The molecular size of the recombinant proteins were determined using Biosep Sec– S4000 column (Phenomenex) calibrated using known protein standards. To study the interaction between αA and αB crystallin, 50 µg of 6H–αA was treated with varied concentrations (0 – 150 µg) of labeled αB, the volume was adjusted to 250 µl using binding buffer (50mM phosphate buffer, 300mM NaCl, and 10 mM imidazole, pH 7.5) and the tubes were incubated at 37oC to allow the subunits to exchange. After 4 hr, 50 µl of Ni–NTA magnetic agarose beads (5% slurry) (Qiagen) was added to pull down protein complex. The unbound sample was pipeted out after placing the tubes on a magnetic rack. The gel was washed (250 µl) three times using the binding buffer and the complex was eluted from the gel using 100 µL of 100 mM EDTA. The amount of αB interacting with 6H–αA was determined using a fluorescence spectrophotometer. Results: Both 6H–αA and αB were purified as homo–aggregates with a molecular mass of 580–610 kD. Introduction of 6H tag did not alter the structural properties of αA–crystallin. In absence of αB, the gel used was sufficient to pull down 24.8 µg of 6HαA. The amount of αB pulled down by the magnetic bead as a result of interaction with 6H–αA increased with increasing concentrations of αB in the reaction mixture. At 100 µg concentration of αB, 8.05 µg was found to be interacting with 6H–αA, further increase in αB concentrations in the reaction mixture did not increase its interaction with 6H–αA. Labeled BSA did not bind to the gel or 6H–αA. Conclusions: The results suggest that αA and αB interact at a ratio of 3.08 : 1. The method can be used to isolate and identify proteins that interact with α–crystallins.
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