In vitroselection and evolution of functional proteins by using ribosome display

Abstract

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 10 8 -fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.

Selection of scFvs specific for HBV DNA polymerase using ribosome display

“…Here we performed ribosome display, an in vitro display method, using an in vitro transcription and translation process. These processes, which were based on E. coli and rabbit reticulocyte systems, have been described previously in studies that used ribosome display to express and select functional scFvs (Hanes and Pluckthun, 1997;He and Taussig, 1997). Because the library size of a ribosome display is not limited by transformation, we hypothesized that antigen-specific scFvs would be more easily obtained from DNA libraries using ribosome display than by in vivo display methods.…”

“…The PCR products obtained are then used for the next cycle of enrichment. This method was originally developed as an efficient tool for obtaining scFvs that have binding affinity to target antigen (Hanes and Pluckthun, 1997;He and Taussig, 1997), and some antibodies with high affinity have been selected and evolved from mouse and human libraries using this method (Hanes et al, 1998(Hanes et al, , 2000He et al, 1999). The advantages of ribosome display are that, in comparison to phage display, larger libraries can be constructed without the transformation step, and the libraries can be further diversified by PCR during ribosome display (Dall'Acqua and Carter, 1998).…”

Beyond the Natural Proteome

“…Ribosome display is an in vitro method of selection invented by Plückthun (Hanes & Plückthun, 1997). It was the first in vitro selection method, inspired by work of Mattheakis and co-workers (Mattheakis, Bhatt & Dower, 1994) who demonstrated affinity selection using polysomes which enable the critical link between genotype and phenotype that is essential for any biopanning application.…”

“…Ribosome display also uses the E. coli S30 system to create whole, correctly-folded proteins that remain coupled with the S30 complex and mRNA (Hanes & Plückthun, 1997). In the first instance, PCR is employed to both amplify the library and couple it with a T7 promoter and ribosome-binding site.…”

NMR analysis on the sialic acid‐binding mechanism of an R‐type lectin mutant by natural evolution‐mimicry

“…developed into a high-throughput technology by combination of error-prone PCR with a recently reinforced ribosome display system (originally developed by Hanes et al [18]). Sia-binding lectins were engineered from the C-terminal domain of earthworm 29-kDa Gal-binding lectin (designated EW29Ch) belonging to the R-type lectin family (Pfam00652) [19] as a scaffold protein by natural evolution-mimicry-EW29Ch has a considerable advantage over other proteins in terms of productivity (> 10 mg of soluble protein is obtained from a 1-L culture) [3].…”