Ribosome display

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Ribosome Display is a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand. The process results in translated proteins that are associated with their mRNA progenitor which is used, as a complex, to bind to an immobilized ligand in a selection step. The mRNA-protein hybrids that bind well are then reverse transcribed to cDNA and their sequence amplified via PCR. The end result is a nucleotide sequence that can be used to create tightly binding proteins.

[edit] The process

Ribosome display refers to the protein binders which are non-covalently linked to and generated from sequences of cDNA containing both a transcription promoter sequence and known ends that will be handles for PCR. This cDNA is created and then transcribed into mRNA. tRNA and ribosomes are added which proceeds to translate the mRNA into a protein until it reaches a 3' unnatural base which causes the ribosome to pause. The temperature is then lowered and salt is added to make the mRNA-ribosome-translated protein complex more stable. The whole complex is then used in the binding selection step.

The next step in the process of ribosome display is to start selecting for things worth keeping. The selection step typically involves an affinity chromatography column with a resin bed containing the ligands you wish the protein to bind to. The binders are run onto the column and those that bind well are immobilized. Subsequent elution of the binders via high salt concentrations or mobile ligands which complex with the binding motif of the protein allowing dissociation of the mRNA. The mRNA can then be reverse transcribed back into cDNA, could be mutated via mutagenesis methods, and fed through the process with greater selective pressure to isolate even better binders.

[edit] Advantages of ribosome display

By having the protein progenitor attached to the complex, the processes of ribosome display skips the microarray/peptide bead/multiple-well sequence separation that is common in assays involving nucleotide hybridization and provides a ready way to amplify the proteins that do bind without decrypting the sequence until necessary. At the same time, this method relies on generating large, concentrated pools of sequence diversity without gaps and keeping these sequences from degrading, hybridizing, and reacting with each other in ways that would create sequence-space gaps.

Competing methods for protein evolution in vitro are phage display, yeast display, bacterial display, and mRNA display.Ribosome display is an in vitro selection and evolution technology for proteins and peptides from large libraries1. As it is performed entirely in vitro, there are two main advantages over other selection technologies2, 3. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations (Box 1). A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide (Fig. 1). The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for analysis. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 extract for in vitro translation, based on the work originally described and further refined in our laboratory1. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display4, 6, 7 is also included in this issue8.