Introduction Directed evolution technologies of proteins has beendeveloping for many decades for the purpose of desired properties or otherspecific characteristicsJD1 . Differentmethods of library screening have been found to identify the desired variants, andthere aretwo broad categories of methods: screening and selectionJD2 . Amongselection methods, display technologies are widely used as a high-throughputselection tool since their expressed protein can easily access to the external environmentJD3 , thereforeallows quick enrichment of target protein. Two types ofdisplay technologies can be distinguished based on the use of living cells orcell extracts. In vivo approachesinclude some widely used technologies such as yeast two-hybrid system 1, cellsurface display 2, phage display 3 and invivo compartmentalization.
In vitro approachesinclude ribosome display, mRNA/cDNA display, and in vitro compartmentalization. For in vivo technologies, some merits appear in the selection, e.g, lownon-specific background in cell surface display by using FACS 4, compatibilitywith protein crossing membranesJD4 , andrelative simplicity in performance. However, the diversity of primary librariesis limited by the efficiency of transformation and transfection when geneinformation is introduced into cells.
As to fully in vitro technologies, the upper limit of the library size isdictated by the genetic material (the amount of synthesized DNA, the volume ofPCR). Oppositeto the highly regulated translation performed in cells, in vitro technologies can be easily combined with PCR-basedrandomization techniques, which further increase the library diversityJD5 5. This review JD6 willintroduce the mechanism as well as the methodology of mRNA display and ribosomedisplay, which are frequently used for invitro directed evolution, and concisely summarize their development andapplication. At last, some improvements on mRNA display and cDNA display willbe mentioned.
1 In vitro directed evolution technologiesJD7 1.1 Ribosome DisplayRibosomedisplay is an in vitro displaytechnology used for selection of proteins. In ribosome display, after translation ribosome stalls at the end ofmRNA which lacks stop codon, with transcribed protein connected to thepeptidyl-tRNA by an ester bond, and thus form themRNA-ribosome-protein complex. Without stop codon, release factors cannot bindto the mRNA and initiate peptide release. The principle of ribosome display isillustrated at Figure 1. FormedmRNA-ribosome-protein ternary complex is used for affinity selection. After selection,the mRNA of enriched proteins is recovered, reverse transcribed and amplifiedby PCR to form the library for next round of selection.
A successful ribosomedisplay selection relies on some essential points. It is necessary for thetemplate to contain T7 promoter, 5′-stemloop, ribosome binding site, regulatorysequence for translation, DNA fragment and spacer containing a region of 3′-stemloop(Figure 2). Thepresence of stemloops is important for resistance to RNase which acts on the 5′-endand 3′-end of mRNA during in vitrotranslation.
A notable increase of efficiency was observed when stemloops wereintroduced 27. The spacer region is crucial for proper protein foldingbecause its translated part fills the tunnel of ribosome, thus providing somedistance and flexibility for the target protein to fold. Second, the antisenseoligonucleotides oftransfer-messenger RNA JD8 need to beadded to prevent the rescue of stalled ribosome 28. Third, in the case of lowmRNA recovery after selection, extra attention is needed to keep mRNA fromdegradation. Ribosomedisplay shares the same advantages with other in vitro display technologies like mRNA display in terms of largelibrary size because of its inherent property JD9 and highlydiverse library achieved by combination of PCR-based randomization techniques.
Another advantage is that after selection of the target, only the mRNA is requiredfor subsequent use, which can be released just by adding EDTA. However, mildconditions are required in ribosome display because of a relatively weaklinkage between genotype and phenotype. Ribosome display was first reportedin 1997, for the selection of single chain fragments of an antibody 29. Thesystem was improved soon after with respect to the folding efficiency of scFvfragments 30. The ability of evolving the whole protein from scratch JD10 was first demonstrated by selecting antibodies forimproved affinity, and by adding additional diversity through randommutagenesis 31. Many works on antibody selection have been reported 32 33and recently the translation and purification procedures in selection of singledomain antibody using ribosome display were optimized 34. Differenttranslation systems such as eukaryotic cell-free translation systems 35 36and PURE system 37 have also been applied to ribosome display.
1.2 mRNA displaymRNA display is an entire JD11 in vitro displaytechnology in which, different from ribosome display, phenotype molecule(peptide/protein) and genotype molecule (mRNA that encoded it) are boundtogether via a covalent linkage. The key to this technology is puromycin, anantibiotic serving as protein synthesis inhibitor, introduced on the 3′-end ofthe mRNA transcribed from a DNA library.
Puromycin contains an analog of tyrosine linked through amidebond to the 3′ position of a modified adenosine (Figure 3), which mimics the 3′-endof tyrosyl-tRNA. At the end of the transcription, attached puromycin enters theA site of ribosome and gets transferred to the growing peptide chain, stallsthe ribosome and forms a covalent bond between mRNA and a correspondingpeptide. The typical scheme of a single round of mRNA display selection is showedin Figure 4. Briefly, a given DNA library is converted to the mRNA librarythrough transcription, then ligated to a length ofsynthetic oligonucleotides with puromycin at 3′ end. After in vitro translation, mRNA and corresponding protein are covalentlybind together, and the pool of mRNA/protein fusion is subjected to affinitybinding of the target of interest.
Finally, reverse transcription and PCR areperformed to recover and enrich the cDNA of the bound protein, being used asinput library for next round of selection. Among all the selection technologieswhich have been widely used, mRNA display possesses several advantages. Thefirst is the ability to process largelibrary size. The formation of the peptide in mRNA display relies oncell-free translation, which eliminates the limitation of library size resultfrom transformation and transfection in cell-surface displays and phagedisplay. The library size of cell-based selections, such as the yeasttwo-hybrid system, bacteria and yeast surface display, is typically limited toapproximately 106 6.
In phage display, the size of library reachesaround 108 7. While for mRNA display, the library size is onlylimited by the amount of in vitrotranslation mixture being used and can reach 1012 -1014sequences 8. The second is highfidelity during selection. With every given mRNA, only single copy of a peptideis displayed. Accordingly, the enrichment of sequences is based solely on theaffinity of the corresponding peptide towards its target.
On the other hand,multiple copies of one peptide displaying on the surface of phage and cell giverise to the enrichment of peptide with weak target affinity due to avidityeffect 9. The third is the efficientsynthesis of the enriched peptide. Unlike the in vivo translation, cell-free translation methods have beendeveloped for higher quality protein synthesis and broader use. With lownuclease and protease activity, some reconstituted systems comprising ofpurified components can facilitate the full-length peptide synthesis 10 andresult in an easy purification 11.
mRNA display with reconstituted E. coli ribosomal translation systemalso enables the synthesis of unnatural peptides 12 13. The fourth is thepossibility of using stringent selectionconditions to minimize the possibility of nonspecific sequences beingselected. By contrast, the selection conditions of some cellular approaches arerestricted to keep the cell integrity. The first originalwork on mRNA display dates back to 1997 14 15, where the basic selection schemewas developed. After that, mRNA display was used to select peptides from alibrary of randomized linear peptide 16 17 and to select antibody 18 aswell as antibody mimics 19.
Meanwhile, optimization in terms of library size20 and displayed protein size 21 improved diversity and efficiency of theselection system. The first application of mRNA display targetingthe selection of an enzyme was done in 2007 24. Seelig’s group established the general scheme (Figure 5) for directselection of enzymes catalyzing bond-forming reactions. A primer bearingsubstrate A (5′-triphosphate-activated RNA) was designed for reversetranscription.
Followed by reverse transcription and incubation with substrateB (biotinylated oligonucleotides), displayed protein with catalytic activitycatalyzed the ligation between substrates A and B and covalently linked them tomRNA/cDNA-protein fusion. The ligated products were captured on streptavidinbeads, in which cDNA was amplified for next round of selection. In-betweenmultiple rounds of selection, mutagenesis, and error-prone PCR were performedto increase the population of ligase variants. According to the analysis ofsequence, characterization and reaction rate enhancement, they demonstratedthat genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information byselection from an initial protein library of very high diversity with productformation as the sole selection criterion.
Based on the above, a detailedgeneral protocol for directed evolution of ligase was set 25 and improved26. Overall, significant genetic diversity and intrinsic high throughput makemRNA display selection a powerful tool for protein directed evolution.1.3 cDNA displayVarious optimizations and improvements have been developed tomake mRNA display more powerful, promising and efficient since it has beencreated. To address the problems occurred due to the vulnerability of mRNA molecule,cDNA display, a variation of mRNA display, was found and first reported in 200938. The key point of this method is the design of a novel puromycin linker(Figure 6).
The linker contains ligationsite, biotin site, reverse transcription primer site and restriction enzymesite, which enables rapid ligation of mRNA and linker,biotin/streptavidin-based purification, and cDNA synthesis by reversetranscription, meanwhile prevents degradation of mRNA. In the first study of cDNA display, researchers chose an affinityscreening based on the highly specific interaction between BDA (B domain ofprotein A) and IgG to evaluate the validity of screening a target moleculeusing cDNA display. They designed a mixed pool comprises equimolar ratio ofcDNA displayed BDA and PDO (act as non-target) and performed one round ofscreening on the mixed pool against IgG. The result showed that 20 fold higheramount of BDA molecules were selected out of mixed pool than which of PDOmolecules.
The validity of cDNA display hasbeen proved though, some research still needed to be done to improve thismethod. In order to resolve the problem that the productivity ofcDNA-protein fusion turned out to be very limited (0.1% of the initial mRNAs),a study has been done to investigate the reason of the low yield and regulatedsome conditions like reducing buffer exchange for His-tag purification andincreasing the amount of SA beads 39. Recently an optimized puromycin linker40 and a photo-cross-linker 41 for cDNA display were reported.
In terms ofin vitro selection speed, by integrating transcription and translation into onstep and skipping the ligation between mRNA and puromycin-linker, 6 rounds ofselections can be performed within 14 h, making the display selection less time-consuming42.1.4 IVC (in vitro compartmentalization)Although different from natural compartments, like bacteriaand yeast, artificial compartments can also serve the purpose of coupling geneand its encoded protein in separated space. Water-in-oil (W/O) emulsiondroplets have been used as such man-made compartments to allow fortranscription and translation of individual gene proceed separately 43.
Normally the water-in-oil emulsion is prepared by stirring anaqueous solution containing a library of genes and in vitro expression systeminto an oil-surfactant mixture. After transcription and translation, genes canbe associated with gene products through covalent linkage or microbeads. Thenthe emulsion is broken and selection is performed.
Finally, the target gene isenriched through PCR. IVC has two advantages in terms of enzyme evolution. Exceptbeing capable of processing large library size (108–1011genes), IVC can select for more enzyme properties, such as regulatory andcatalytic activity, than just binding activity.
Moreover, it allows forselection of enzyme with multiple turnover. There are still some limitationsexist during screening which researchers have been working on. Griffths et alfirst combined water-in-oil-in-water double emulsion and FACS system togetherfor directed evolution of b-galactosidase and got a product sorting rate of20000 droplets s-144. However, some limitations, like thepolydispersity of droplets, were noticed. To get rid of those limitations theydeveloped a droplet-based microfluidic system instead of FACS, which resultedin an order of magnitude lower polydispersity than FACS system at a cost of10-fold lower screening speed45. Some other study also applied microfluidicplatform, for example screening for activity of FeFe hydrogenase46, hydrolyticactivities of a promiscuous sulfatase47 and glucose oxidase activity. On theother hand, other studies focused on some improvements when using FACS systemfor screening, like a generalizable protocol of producing monodispersepicolitre double emulsion droplets for directed evolution48 and a protocol employingmembrane-extrusion technique to generate uniform emulsion droplets49.
JD1? Needs rephrasing, not clear. JD2????? JD3??Don’t understand this. JD4?Is this an advantage? JD5Howdoes translation compare with gene randomization? JD6Review? JD7Whatyou describe under this title are different genotype-phenotype linkages(display methods) which are used for in vitro directed evolution. The title ismuch broader than the contents and needs revision. Display is just one part ofthe directed evolution process. JD8? JD9Whatproperty? JD10Whatdoes this mean? JD11Doesthis mean the previous one is not?