Refolding of firefly luciferase immobilized on agarose beads

The renaturation yield of the denatured firefly luciferase decreased strong ly with increasing protein concentration in a renaturation buffer, because of aggregation, In this study, firefly luciferase was immobilized on agaros e beads at a high concentration, Although the protein concentration was ext remely high (about 100-fold) compared to that of soluble luciferase, the re naturation yield was comparable with that for the soluble one. Thus, immobi lization was shown to be effective for avoiding aggregation of firefly luci ferase. It was also shown that the optimum buffer conditions for renaturati on of the immobilized luciferase were the same as those for the renaturatio n in solution, Also, it was indicated that electrostatic interactions betwe en a protein and the matrix have a negative effect on renaturation of the i mmobilized luciferase since the renaturation yield decreased at acidic pH o nly for the immobilized luciferase, These novel observations are described in detail in this paper.