Selection of catalytically active biotin ligase and trypsin mutants by phage display |
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Authors: | Heinis Christian; Huber Adrian; Demartis Salvatore; Bertschinger Julian; Melkko Samu; Lozzi Luisa; Neri Paolo; Neri Dario |
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Affiliation: | 1 Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland and
2 Dipartimento di Biologia Molecolare, Sez. di Chimica Biologica, Università di Siena, Via Fiorentina 1, 53100 Siena, Italy |
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Abstract: | Phage display has been shown to facilitate greatly the selectionof polypeptides with desired properties by establishing a directlink between the polypeptide and the gene that encodes it. However,selection for catalytic activities displayed on phage remainsa challenge, since reaction products diffuse away from the enzymeand make it difficult to recover catalytically active phageenzymes.We have recently described a selection methodology in whichthe reaction substrate (and eventually the reaction product)is anchored on calmodulin-tagged phageenzymes by meansof a calmodulin binding peptide. Phage displaying a catalyticactivity are physically isolated by means of affinity reagentsspecific for the product of reaction. In this study, we investigatedthe efficiency of selection for catalysis by phage display,using a ligase (the Escherichia coli biotin ligase BirA) andan endopeptidase (the rat trypsin His57 |
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