Libraries of random-sequence polypeptides produced with high yield as carboxy-terminal fusions with ubiquitin.
Libraries of random-sequence polypeptides have been shown to be valuable 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 limited to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypeptide length. Cloned as carboxy-terminal extensions of ubiquitin, the fusions were produced in E. coli at high levels, and were purified to homogeneity. The majority of the extension proteins examined could be cleaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. The libraries described here are appropriate sources of novel polypeptides with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward the latter goal, we have examined structural properties of random-sequence proteins purified from these libraries. Quite surprisingly, fluorescence emission spectra of intrinsic tryptophan residues in several purified fusion proteins, under native-like and denaturing conditions, often resemble those expected for folded and unfolded states, respectively. The results presented here detail an important expansion in the range of potential uses for random-sequence polypeptide libraries.
LaBean
T H
TH
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104, USA.
Kauffman
S A
SA
Butt
T R
TR
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010452:Peptide Biosynthesis; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014452:Ubiquitins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins