Isolating substrates for an engineered alpha-lytic protease by phage display.
Panning of a substrate phage library with an alpha-lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered alpha-lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P4 positions, whereas Asp dominated the putative P1 position.
Lien
Samantha
S
Department of Biochemistry, University of Adelaide, South Australia 5005, Australia. slien@gene.com
Francis
Geoffrey L
GL
Graham
Lloyd D
LD
Wallace
John C
JC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry / Q000235:genetics; D019921:Biotinylation; D002352:Carrier Proteins / Q000737:chemistry; D005822:Genetic Vectors; D007700:Kinetics; D009154:Mutation; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry; D019151:Peptide Library; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry
D002352:Carrier Proteins; C024013:DNA replication complex protein, Bacteriophage lambda; D009842:Oligopeptides; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C108671:biotin carboxyl carrier protein (70-156); D012697:Serine Endopeptidases; D013381:Subtilisins; C021826:myxobacter alpha-lytic proteinase