LIBRARIES OF RANDOM-SEQUENCE POLYPEPTIDES PRODUCED WITH HIGH-YIELD ASCARBOXY-TERMINAL FUSIONS WITH UBIQUITIN

Libraries of random-sequence polypeptides have been shown to be valuab le sources of novel molecules possessing a variety of useful biologic- like activities, some of which may hold promise as potential vaccines and therapeutics. Previous random peptide expression systems were limi ted to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypept ide length. Cloned as carboxy-terminal extensions of ubiquitin, the fu sions were produced in E. coli at high levels, and were purified to ho mogeneity. The majority of the extension proteins examined could be cl eaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. T he libraries described here are appropriate sources of novel polypepti des with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward th e latter goal, we have examined structural properties of random-sequen ce proteins purified from these libraries. Quite surprisingly, fluores cence emission spectra of intrinsic tryptophan residues in several pur ified fusion proteins, under native-like and denaturing conditions, of ten resemble those expected for folded and unfolded states, respective ly. The results presented here detail an important expansion in the ra nge of potential uses for random-sequence polypeptide libraries.