A SPARSE-MATRIX SCREEN TO ESTABLISH INITIAL CONDITIONS FOR PROTEIN RENATURATION

Protein renaturation is of particular interest not only for the basic mechanisms of protein folding but also as a practical problem for prot eins overexpressed in microorganisms, since recombinant proteins may a ccumulate as misfolded aggregates in ''inclusion bodies'' that are ina ctive after purification. We have established a systematic screening m ethod to identify conditions which promote protein renaturation. A mat rix of 50 different buffers, which were originally developed for prote in crystallization, were found to facilitate the renaturation for eigh t of nine different proteins examined. The proteins tested include the adhesive protein bindin, recombinant bindin, and a variety of enzymes , including bacterial alkaline phophatase, horseradish peroxidase, lys ozyme, trypsin, beta-galactosidase, rabbit carboxylesterase, and acety lcholinesterase. The total amount of activity recovered varied hom 9 t o 333% depending on the protein. The conditions that were found to pro mote renaturation are very different from the optimal conditions for e nzyme activity. The finding that most of the proteins tested renatured to a significant extent in one or more of the buffers in the matrix s uggests that the sparse matrix screen may be of general utility for es tablishing initial renaturation conditions for a wide variety of prote ins. Once initial renaturation conditions have been identified, the co nditions may be optimized by systematically altering other parameters of the renaturation process. (C) 1995 Academic Press, Inc.