Activity-based selection of a proteolytic species using ribosome display.
We have examined the potential of displaying a protease species in vitro using ribosome display and demonstrate specific capture on the basis of its catalytic activity. Using a model bacterial cysteine protease, sortase A (SrtA), we show that this enzyme can be functionally expressed in vitro. By overlap PCR we constructed ribosome display templates with the SrtA open reading frame fused to a C terminal glycine-serine rich flexible linker and a tether derived from eGFP. Using the broad range cysteine protease irreversible inhibitor E-64 linked to acrylic beads, we show that we can isolate SrtA ribosome display ternary complexes, and recover their encoding mRNA by RT-PCR. This recovery was lost when applied to a SrtA catalytically inactive mutant, or could be alleviated by competition with free inhibitor. This sensitive technique could be further developed to allow the screening of proteases against putative inhibitors and/or the identification of novel proteolytic species.
Quinn
Derek J
DJ
Molecular Therapeutics, School of Pharmacy, Queen's University of Belfast, 97 Lisburn Road, Belfast, Antrim BT9 7BL, UK.
Cunningham
Stephen
S
Walker
Brian
B
Scott
Christopher J
CJ
D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008722:Methods; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000201:enzymology*
D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; D012333:RNA, Messenger; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D007930:Leucine; C024974:E 64