In Vitro Selection for Catalytic Activity with Ribosome Display

Abstract

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.

Insights into Positive and Negative Requirements for Protein–Protein Interactions by Crystallographic Analysis of the β-Lactamase Inhibitory Proteins BLIP, BLIP-I, and BLP

“…The originally identified BLIP (from Streptomyces clavuligerus) 9 has become a model for a broad range of structural, mutagenic, biophysical, and computational studies. [10][11][12][13][14][15][16][17][18][19][20][21] BLIP has two close probable homologues: BLIP-I (from Streptomyces exfoliatus) and β-lactamase-inhibitoryprotein-like protein (BLP; S. clavuligerus). No other related protein † is found in any Streptomyces genome that has been sequenced fully ‡ or partially §.…”

Identification of a Functional Epitope of the Nogo Receptor by a Combinatorial Approach Using Ribosome Display

“…The solution was incubated with 100 ml butylSepharose for one hour at 4 8C and the supernatant containing the ternary complexes was then applied to a gel filtration column to exchange the buffer to WBKT. 43 Selection of binders to Nogo-66 was performed for one hour at 4 8C by adding half of the eluted ternary complexes to 10 7 paramagnetic beads coated with either pD-Nogo-66 or Trx-Nogo-66 in the presence of BSA (final concentration 5 mg/ml) and Saccharomyces cerevisiae RNA (final concentration 250 mg/ml). After washing, RNA elution and purification, reverse transcription (RT) was performed with the Thermoscript-Kit (Invitrogen) and primer rdN2_L (5 0 -GGCGCGGTTGCGGTACCCATAG-3 0 ), including a RNaseH digestion step after the RT reaction.…”

Separation methods applicable to the evaluation of enzyme–inhibitor and enzyme–substrate interactions

“…Retrieval of the complexes with the mechanism-based inhibitor, ampicillin sulfone, immobilized on magnetic beads provided a simple method to separate active conjugates. Testing ribosome display with an entire ␤-lactamase library is pending, but initial results of 100-fold per cycle enrichment factors indicate that this method may be an attractive system for future development of novel ␤-lactamase enzymes [91,92].…”