title: Selection of catalytically active biotin ligase and trypsin mutants by phage display.
abstract: Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.
authors.affiliation: Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Heinis
authors.forename: A
authors.initials: A
authors.lastname: Huber
authors.forename: S
authors.initials: S
authors.lastname: Demartis
authors.forename: J
authors.initials: J
authors.lastname: Bertschinger
authors.forename: S
authors.initials: S
authors.lastname: Melkko
authors.forename: L
authors.initials: L
authors.lastname: Lozzi
authors.forename: P
authors.initials: P
authors.lastname: Neri
authors.forename: D
authors.initials: D
authors.lastname: Neri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D002147:Calmodulin; D019731:Carbon-Nitrogen Ligases / Q000032:analysis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D051381:Rats; D012097:Repressor Proteins; D014157:Transcription Factors; D014357:Trypsin / Q000032:analysis / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002147:Calmodulin; D029968:Escherichia coli Proteins; D019151:Peptide Library; D012097:Repressor Proteins; D014157:Transcription Factors; D014357:Trypsin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli