title: Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.
abstract: We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery.
authors.affiliation: Janusys Corporation, Saitama Industrial Technology Center, Skip City, Kawaguchi, Saitama 333-0844, Japan. naimuddin@janusys.co.jp
authors.forename: Mohammed
authors.initials: M
authors.lastname: Naimuddin
authors.forename: Tai
authors.initials: T
authors.lastname: Kubo
mesh_terms: D000595:Amino Acid Sequence; D002474:Cell-Free System; D005591:Chemical Fractionation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism; D011691:Puromycin / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D004220:Disulfides; D009498:Neurotoxins; D009841:Oligonucleotides; D019151:Peptide Library; D011506:Proteins; D012333:RNA, Messenger; D011691:Puromycin; D019704:Protein Disulfide-Isomerases