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.

Figure 1

Changes in the streamlined mRNA display protocol. A traditional mRNA display protocol is shown,^,, with our modifications highlighted in red: (a) 5’ UTR with T7 promoter and Shine-Dalgarno RBS; (b) mRNA purification with LiCl precipitation instead of lengthy PAGE purification; (c) mRNA-DNA-puromycin purification with ultrafiltration instead of lengthy PAGE purification; (d) in vitro translation with minimal, reconstituted translation system (PURExpress) instead of crude lysate to achieve higher library diversity with less nuclease and protease activity, which, in turn, can (e) eliminate need for purification of selection particles; (f) postpone reverse transcription, thus eliminating an additional purification step as well as avoiding a high-temperature incubation step of the displayed polypeptide prior to selection.

Figure 2

Model DARPin selections using our streamlined mRNA display protocol. Four binary libraries containing mRNAs encoding H10-2-G3 (H: a Her2 binder) and Off7 (O: a MBP binder) at different molar ratios (1:10, 1:100, 1:1,000, and 1:10,000) were subjected to two to three rounds of selections against immobilized Her2. The original library (Ori.) and selective enrichment of H10-2-G3 in the RT-PCR product after each round of mRNA display (R1, R2, and R3) is visualized by agarose gel electrophoresis. Each selection round took two days in the laboratory.