Directed evolution of chaperone -like activity in small heat shock proteins

Small heat shock proteins (sHSP) have chaperone-like activity as defined by stabilization and refolding of proteins during cell stress and by binding to proteins in their non-native forms. The goal of our studies was to engineer a better sHSP. In order to do this we have developed an in vivo assay to measure chaperone-like activity. This assay is based upon folding of GFP protein which does not express any fluorescence capabilities unless it is folded into its normal tertiary structure. The in vivo assay is easy to use for screening of mutants and chimeric proteins. Improved sHSPs were generated using a new chimeragenesis technique, called h&barbelow;omology i&barbelow;ndependent t&barbelow;argeted chimera production (HITCh). Three genes, alphaA-crystallin, alphaB-crystallin, and the small heat shock protein gene from Methanobacterium janaschii (MjHSP16.5) were used as a basis gene set for testing the technique. Chimeric genes were made and some of the resulting proteins have been found to have greater chaperone-like activity than any of the parental genes after only a single cycle of chimeragenesis. A number of human disease conditions have been correlated with the activity of sHSPs, particularly with the chaperone-like activity. The chaperone-like activity is based upon in vitro protection assays utilizing one of several different protein aggregation or enzyme activity measurements. We have made several of these sHSP mutants utilizing a new site directed mutagenesis technique, INSULT. Our mutants were studied in the in vivo assay for comparison to the in vitro activity measurements previously reported. Our data suggest that in vitro activities should be interpreted with caution. Several mutants reported to have decreased chaperone-like activity have been found to have increased chaperone-like activity when tested in vivo. These studies have proven the versatility of our in vivo assay in testing for chaperone-like activity and served as a platform for development and testing of new chimeragenesis and site-directed mutagenesis techniques in engineering of small heat shock proteins.