Analysis by phage display selection and site-directed retromutagenesis of the Mustard Trypsin Inhibitor 2 reactive site.
The Mustard Trypsin Inhibitor (MSI) family is a small family of plant protease inhibitors so far only found in Brassicaceae. Using a phage display selection, MTI-2 (Mustard Trypsin Inhibitor 2) mutants were detected and analysed for their biochemical characteristics. Retromutants of the selected MTI-2 proteins were constructed and expressed in the Pichia pastoris system. The recombinant proteins were analysed by activity assays against bovine trypsin and Helicoverpa zea trypsin, and by circular dichroism. These analyses suggest a strict requirement for a specific proline residue adjacent to the inhibitor reactive site and give additional insights for future phage display application.
Volpicella
Mariateresa
M
Dipartimento di Biochimica e Biologia Molecolare, University of Bari "A. Moro", Via Amendola 165/A, 70126 Bari, Italy. m.volpicella@biologia.uniba.it
Leoni
Claudia
C
Arnesano
Fabio
F
Gallerani
Raffaele
R
Ceci
Luigi R
LR
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D017343:Genes, Plant; D007313:Insecta / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
C405940:MTI2 protein, Sinapis alba; D019151:Peptide Library; D010940:Plant Proteins; D010447:Peptide Hydrolases; D014357:Trypsin