Selecting folded proteins from a library of secondary structural elements.
A protein evolution strategy is described by which double-stranded DNA fragments encoding defined Escherichia coli protein secondary structural elements (alpha-helices, beta-strands, and loops) are assembled semirandomly into sequences comprised of as many as 800 amino acid residues. A library of novel polypeptides generated from this system was inserted into an enhanced green fluorescent protein (EGFP) fusion vector. Library members were screened by fluorescence activated cell sorting (FACS) to identify those polypeptides that fold into soluble, stable structures in vivo that comprised a subset of shorter sequences ( approximately 60 to 100 residues) from the semirandom sequence library. Approximately 108 clones were screened by FACS, a set of 1149 high fluorescence colonies were characterized by dPCR, and four soluble clones with varying amounts of secondary structure were identified. One of these is highly homologous to a domain of aspartate racemase from a marine bacterium (Polaromonas sp.) but is not homologous to any E. coli protein sequence. Several other selected polypeptides have no global sequence homology to any known protein but show significant alpha-helical content, limited dispersion in 1D nuclear magnetic resonance spectra, pH sensitive ANS binding and reversible folding into soluble structures. These results demonstrate that this strategy can generate novel polypeptide sequences containing secondary structure.
Graziano
James J
JJ
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Liu
Wenshe
W
Perera
Roshan
R
Geierstanger
Bernhard H
BH
Lesley
Scott A
SA
Schultz
Peter G
PG
D019020:Directed Molecular Evolution; D029968:Escherichia coli Proteins; D010446:Peptide Fragments / Q000737:chemistry*; D019151:Peptide Library; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis*; D012995:Solubility
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins