In vitro selection for catalytic activity with ribosome display.
We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.
Amstutz
Patrick
P
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, Switzerland.
Pelletier
Joelle N
JN
Guggisberg
Armin
A
Jermutus
Lutz
L
Cesaro-Tadic
Sandro
S
Zahnd
Christian
C
Plückthun
Andreas
A
D000667:Ampicillin / Q000494:pharmacology; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D002384:Catalysis; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D012270:Ribosomes / Q000378:metabolism*; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D001710:Biotin; D000667:Ampicillin; D001618:beta-Lactamases