Streamlined protocol for mRNA display

Pamela A. Barendt, Daphne T.W. Ng, Casey N. McQuade, Casim A. Sarkar

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

mRNA display is a powerful method for in vitro directed evolution of polypeptides, but its time-consuming, technically demanding nature has hindered its widespread use. We present a streamlined protocol in which lengthy mRNA purification steps are replaced with faster precipitation and ultrafiltration alternatives; additionally, other purification steps are entirely eliminated by using a reconstituted translation system and by performing reverse transcription after selection, which also protects input polypeptides from thermal denaturation. We tested this procedure by performing affinity selection against Her2 using binary libraries containing a nonspecific designed ankyrin repeat protein (DARPin) doped with a Her2-binding DARPin (dopant fraction ranging from 1:10 to 1:10 000). The Her2-binding DARPin was recovered in all cases, with an enrichment factor of up to 2 orders of magnitude per selection round. The time required for 1 round is reduced from ∼4-7 days to 2 days with our protocol, thus simplifying and accelerating mRNA display experiments.

Original languageEnglish (US)
Pages (from-to)77-81
Number of pages5
JournalACS Combinatorial Science
Volume15
Issue number2
DOIs
StatePublished - Feb 11 2013
Externally publishedYes

Keywords

  • binary library
  • directed evolution
  • in vitro translation
  • mRNA display
  • purification

Fingerprint

Dive into the research topics of 'Streamlined protocol for mRNA display'. Together they form a unique fingerprint.

Cite this