Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold.
A disulfide bridge-constrained cellulose binding domain (CBD(WT)) derived from the cellobiohydrolase Cel7A from Trichoderma reesei has been investigated for use in scaffold engineering to obtain novel binding proteins. The gene encoding the wild-type 36 aa CBD(WT) domain was first inserted into a phagemid vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein with retained cellulose binding activity. A combinatorial library comprising 46 million variants of the CBD domain was constructed through randomization of 11 positions located at the domain surface and distributed over three separate beta-sheets of the domain. Using the enzyme porcine alpha-amylase (PPA) as target in biopannings, two CBD variants showing selective binding to the enzyme were characterized. Reduction and iodoacetamide blocking of cysteine residues in selected CBD variants resulted in a loss of binding activity, indicating a conformation dependent binding. Interestingly, further studies showed that the selected CBD variants were capable of competing with the binding of the amylase inhibitor acarbose to the enzyme. In addition, the enzyme activity could be partially inhibited by addition of soluble protein, suggesting that the selected CBD variants bind to the active site of the enzyme.
Lehtiö
J
J
Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Teeri
T T
TT
Nygren
P A
PA
D020909:Acarbose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D020650:Combinatorial Chemistry Techniques; D017912:Inovirus; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library; D013552:Swine; D014242:Trichoderma / Q000201:enzymology; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D010446:Peptide Fragments; D019151:Peptide Library; D002482:Cellulose; D000516:alpha-Amylases; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D020909:Acarbose