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Investigation of de novo Totally Random Biosequences, Part I
Author(s) -
Chiarabelli Cristiano,
Vrijbloed Jan W.,
Thomas Richard M.,
Luisi Pier Luigi
Publication year - 2006
Publication title -
chemistry and biodiversity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 70
eISSN - 1612-1880
pISSN - 1612-1872
DOI - 10.1002/cbdv.200690087
Subject(s) - proteolysis , folding (dsp implementation) , phage display , sequence (biology) , computational biology , chemistry , tripeptide , plasmid , phagemid , circular dichroism , biochemistry , protein tertiary structure , gene , bacteriophage , biology , amino acid , peptide , enzyme , escherichia coli , engineering , electrical engineering
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.