Investigation of de novo totally random biosequences, Part I: A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage.
This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.
Chiarabelli
Cristiano
C
Centro Studi e Ricerche E. Fermi, Via Panisperna 89A, IT-00184 Rome.
Vrijbloed
Jan W
JW
Thomas
Richard M
RM
Luisi
Pier Luigi
PL
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019295:Computational Biology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism
D010455:Peptides; D013917:Thrombin