title: Streamlined protocol for mRNA display.
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.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd Street, Philadelphia, Pennsylvania 19104-6321, United States.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Barendt
authors.forename: Daphne T W
authors.initials: DT
authors.lastname: Ng
authors.forename: Casey N
authors.initials: CN
authors.lastname: McQuade
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
mesh_terms: D017089:Ankyrin Repeat; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D013696:Temperature
chemical_list: D010455:Peptides; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases