Technique review Ribosome display: Cell-free protein display technology

Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein–ribosome–mRNA (PRM) complexes. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression. Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation. It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions. In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution). Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features. In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed. INTRODUCTION Display systems are used for the selection of coding elements (DNA or RNA) from libraries in which the individual peptides or proteins as phenotypes are physically associated with their genetic material. They can also be used to alter the properties of the phenotype by evolution through cycles of mutation, selection and replication. The success of display selection relies on the ability to retrieve the genetic information along with the functional protein. Several methods have been devised and validated, both cell based, such as phage display and cell surface display, and cell free, such as ribosome display and mRNA display. Ribosome display is carried out fully in vitro, which overcomes some of the limitations of cell-based display systems. Through the formation of protein– ribosome–mRNA (PRM) complexes in cell-free systems such as Escherichia coli S30 or rabbit reticulocyte, individual nascent proteins are linked with their corresponding mRNA molecules, permitting selection of the genetic material through the functional properties of the protein, usually as a binding reaction. The mRNA can be amplified and recovered as DNA by reverse transcription polymerase chain reaction (RT-PCR) and further cycled, mutated or cloned. In this paper, ribosome display technology, including its applications in protein selection and evolution in vitro, will be reviewed. RIBOSOME DISPLAY: PRINCIPLE AND ADVANTAGES The key feature of ribosome display is the generation of stable PRM complexes via ribosome stalling such that the nascent protein and mRNA remain associated. 2 0 4 & HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 204–212. JULY 2002 Two strategies which have been used are: (i) addition of antibiotics such as rifampicin and chloramphenicol (for prokaryotic ribosomes) or cycloheximide (for eukaryotic ribosomes) to halt translation at random, or (ii) deletion of the stop codon, normally recognised by release factors which trigger detachment of the nascent polypeptide, to stall the ribosome at the 39 end of the mRNA. Ribosome display offers a number of advantages over cell-based methods such as phage or cell surface display. As has often been noted, the efficiency of transformation imposes a restriction on library diversity for cell-based systems, which does not apply in ribosome display. Thus, larger libraries can be screened in each cycle because PCR products are directly utilised as templates, avoiding the need for cloning. PCR libraries with huge potential diversity (.10 members) can be generated easily and the displayed library size is only dependent on the number of functional ribosomes in the reaction, which can be up to 1014=ml. The larger accessible library renders ribosome display a superior opportunity to select rare sequences and high-affinity combining sites. Moreover, using PCR, further diversity can be continuously introduced into the DNA pools after selection, providing an efficient route for protein evolution. In addition, cell-free systems can produce toxic and proteolytically sensitive or unstable proteins for which bacterial expression is often unsuccessful. They also allow modified amino acids such as chemically labelled or unnatural amino acids to be incorporated into the protein at defined positions. Eukaryotic cell-free systems are also capable of a variety of posttranslational modifications, expanding the possibility of displaying functionally relevant proteins. RIBOSOME DISPLAY SYSTEMS Prokaryotic ribosome (polysome) display The first published description of ribosome display was for peptide selection using a coupled E. coli S30 system and termed ‘polysome display’. A synthetic DNA library encoding random peptide sequences was used to generate polysome complexes by adding chloramphenicol to stop translation. Specific complexes displaying interacting peptide epitopes were captured with immobilised antibody by panning on microtitre wells. The trapped polysomes were disrupted with EDTA to release the bound mRNA, which was then converted and amplified into cDNA by RT-PCR. This procedure was subsequently modified to display folded single-chain antibody fragments. Figure 1A outlines the display cycle. The modified prokaryotic method generated PRM complexes through deletion of the 39 terminal stop codon from DNA. It also included a number of additional components, such as protein disulphide isomerase, vanadyl ribonucleoside complexes and anti-ssrA anti-sense oligouncleotide (to prevent the carboxyterminal addition of an 11 amino acid peptide encoded by the ssrA gene), in the translation mixture in order to promote folding of antibody fragments, stabilise mRNA and inhibit the action of ssrA RNA, respectively. To avoid the disruptive effect of dithiothreitol (DTT) on the folding of antibody domains through reduction of disulphide bridges, transcription and translation were performed separately, and the mRNA was introduced into the uncoupled E. coli S30 translation system lacking DTT. Eukaryotic ribosome display A eukaryotic ribosome display system — ARM (antibody–ribosome–mRNA) display — was developed for the selection of functional single-chain antibody fragments in a coupled rabbit reticulocyte lysate system (Figure 1B). Deletion of the stop codon from the PCR fragment was again used to generate eukaryotic PRM complexes in a simple and rapid procedure. ARM complexes were generated by cell-free expression of PCR fragments followed by specific capture on Advantages of ribosome display & HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 204–212. JULY 2002 2 0 5 Ribosome display: Cell-free protein display technology