Introduction

      Directed evolution technologies of proteins has been
developing for many decades for the purpose of desired properties or other
specific characteristicsJD1 . Different
methods of library screening have been found to identify the desired variants, and
there are
two broad categories of methods: screening and selectionJD2 . Among
selection methods, display technologies are widely used as a high-throughput
selection tool since their expressed protein can easily access to the external environmentJD3 , therefore
allows quick enrichment of target protein. Two types of
display technologies can be distinguished based on the use of living cells or
cell extracts. In vivo approaches
include some widely used technologies such as yeast two-hybrid system 1, cell
surface display 2, phage display 3 and in
vivo compartmentalization. In vitro approaches
include ribosome display, mRNA/cDNA display, and in vitro compartmentalization. For in vivo technologies, some merits appear in the selection, e.g, low
non-specific background in cell surface display by using FACS 4, compatibility
with protein crossing membranesJD4 , and
relative simplicity in performance. However, the diversity of primary libraries
is limited by the efficiency of transformation and transfection when gene
information is introduced into cells. As to fully in vitro technologies, the upper limit of the library size is
dictated by the genetic material (the amount of synthesized DNA, the volume of
PCR). Opposite
to the highly regulated translation performed in cells, in vitro technologies can be easily combined with PCR-based
randomization techniques, which further increase the library diversityJD5  5.

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This review JD6 will
introduce the mechanism as well as the methodology of mRNA display and ribosome
display, which are frequently used for in
vitro directed evolution, and concisely summarize their development and
application. At last, some improvements on mRNA display and cDNA display will
be mentioned.

1 In vitro directed evolution technologiesJD7 

1.1  Ribosome Display

Ribosome
display is an in vitro display
technology used for selection of proteins. In ribosome display, after translation ribosome stalls at the end of
mRNA which lacks stop codon, with transcribed protein connected to the
peptidyl-tRNA by an ester bond, and thus form the
mRNA-ribosome-protein complex. Without stop codon, release factors cannot bind
to the mRNA and initiate peptide release. The principle of ribosome display is
illustrated at Figure 1.

Formed
mRNA-ribosome-protein ternary complex is used for affinity selection. After selection,
the mRNA of enriched proteins is recovered, reverse transcribed and amplified
by PCR to form the library for next round of selection. A successful ribosome
display selection relies on some essential points. It is necessary for the
template to contain T7 promoter, 5′-stemloop, ribosome binding site, regulatory
sequence for translation, DNA fragment and spacer containing a region of 3′-stemloop
(Figure 2).

The
presence of stemloops is important for resistance to RNase which acts on the 5′-end
and 3′-end of mRNA during in vitro
translation. A notable increase of efficiency was observed when stemloops were
introduced 27. The spacer region is crucial for proper protein folding
because its translated part fills the tunnel of ribosome, thus providing some
distance and flexibility for the target protein to fold. Second, the antisense
oligonucleotides of
transfer-messenger RNA JD8 need to be
added to prevent the rescue of stalled ribosome 28. Third, in the case of low
mRNA recovery after selection, extra attention is needed to keep mRNA from
degradation.

Ribosome
display shares the same advantages with other in vitro display technologies like mRNA display in terms of large
library size because of its inherent property JD9 and highly
diverse library achieved by combination of PCR-based randomization techniques.
Another advantage is that after selection of the target, only the mRNA is required
for subsequent use, which can be released just by adding EDTA. However, mild
conditions are required in ribosome display because of a relatively weak
linkage between genotype and phenotype. 

     Ribosome display was first reported
in 1997, for the selection of single chain fragments of an antibody 29. The
system was improved soon after with respect to the folding efficiency of scFv
fragments 30. The ability of evolving the whole protein from scratch JD10 was first demonstrated by selecting antibodies for
improved affinity, and by adding additional diversity through random
mutagenesis 31. Many works on antibody selection have been reported 32 33
and recently the translation and purification procedures in selection of single
domain antibody using ribosome display were optimized 34. Different
translation systems such as eukaryotic cell-free translation systems 35 36
and PURE system 37 have also been applied to ribosome display.

1.2  mRNA display

mRNA display is an entire JD11 in vitro display
technology in which, different from ribosome display, phenotype molecule
(peptide/protein) and genotype molecule (mRNA that encoded it) are bound
together via a covalent linkage. The key to this technology is puromycin, an
antibiotic serving as protein synthesis inhibitor, introduced on the 3′-end of
the mRNA transcribed from a DNA library.

Puromycin contains an analog of tyrosine linked through amide
bond to the 3′ position of a modified adenosine (Figure 3), which mimics the 3′-end
of tyrosyl-tRNA. At the end of the transcription, attached puromycin enters the
A site of ribosome and gets transferred to the growing peptide chain, stalls
the ribosome and forms a covalent bond between mRNA and a corresponding
peptide. The typical scheme of a single round of mRNA display selection is showed
in Figure 4.

Briefly, a given DNA library is converted to the mRNA library
through transcription, then ligated to a length of
synthetic oligonucleotides with puromycin at 3′ end. After in vitro translation, mRNA and corresponding protein are covalently
bind together, and the pool of mRNA/protein fusion is subjected to affinity
binding of the target of interest. Finally, reverse transcription and PCR are
performed to recover and enrich the cDNA of the bound protein, being used as
input library for next round of selection. Among all the selection technologies
which have been widely used, mRNA display possesses several advantages. The
first is the ability to process large
library size. The formation of the peptide in mRNA display relies on
cell-free translation, which eliminates the limitation of library size result
from transformation and transfection in cell-surface displays and phage
display. The library size of cell-based selections, such as the yeast
two-hybrid system, bacteria and yeast surface display, is typically limited to
approximately 106 6. In phage display, the size of library reaches
around 108 7. While for mRNA display, the library size is only
limited by the amount of in vitro
translation mixture being used and can reach 1012 -1014
sequences 8. The second is high
fidelity during selection. With every given mRNA, only single copy of a peptide
is displayed. Accordingly, the enrichment of sequences is based solely on the
affinity of the corresponding peptide towards its target. On the other hand,
multiple copies of one peptide displaying on the surface of phage and cell give
rise to the enrichment of peptide with weak target affinity due to avidity
effect 9. The third is the efficient
synthesis of the enriched peptide. Unlike the in vivo translation, cell-free translation methods have been
developed for higher quality protein synthesis and broader use. With low
nuclease and protease activity, some reconstituted systems comprising of
purified components can facilitate the full-length peptide synthesis 10 and
result in an easy purification 11. mRNA display with reconstituted E. coli ribosomal translation system
also enables the synthesis of unnatural peptides 12 13. The fourth is the
possibility of using stringent selection
conditions to minimize the possibility of nonspecific sequences being
selected. By contrast, the selection conditions of some cellular approaches are
restricted to keep the cell integrity.

  The first original
work on mRNA display dates back to 1997 14 15, where the basic selection scheme
was developed. After that, mRNA display was used to select peptides from a
library of randomized linear peptide 16 17 and to select antibody 18 as
well as antibody mimics 19. Meanwhile, optimization in terms of library size
20 and displayed protein size 21 improved diversity and efficiency of the
selection system. The first application of mRNA display targeting
the selection of an enzyme was done in 2007 24.

Seelig’s group established the general scheme (Figure 5) for direct
selection of enzymes catalyzing bond-forming reactions. A primer bearing
substrate A (5′-triphosphate-activated RNA) was designed for reverse
transcription. Followed by reverse transcription and incubation with substrate
B (biotinylated oligonucleotides), displayed protein with catalytic activity
catalyzed the ligation between substrates A and B and covalently linked them to
mRNA/cDNA-protein fusion. The ligated products were captured on streptavidin
beads, in which cDNA was amplified for next round of selection. In-between
multiple rounds of selection, mutagenesis, and error-prone PCR were performed
to increase the population of ligase variants. According to the analysis of
sequence, characterization and reaction rate enhancement, they demonstrated
that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by
selection from an initial protein library of very high diversity with product
formation as the sole selection criterion. Based on the above, a detailed
general protocol for directed evolution of ligase was set 25 and improved
26. Overall, significant genetic diversity and intrinsic high throughput make
mRNA display selection a powerful tool for protein directed evolution.

1.3  cDNA display

Various optimizations and improvements have been developed to
make mRNA display more powerful, promising and efficient since it has been
created. 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 2009
38. The key point of this method is the design of a novel puromycin linker
(Figure 6). The linker contains ligation
site, biotin site, reverse transcription primer site and restriction enzyme
site, which enables rapid ligation of mRNA and linker,
biotin/streptavidin-based purification, and cDNA synthesis by reverse
transcription, meanwhile prevents degradation of mRNA. In the first study of cDNA display, researchers chose an affinity
screening based on the highly specific interaction between BDA (B domain of
protein A) and IgG to evaluate the validity of screening a target molecule
using cDNA display. They designed a mixed pool comprises equimolar ratio of
cDNA displayed BDA and PDO (act as non-target) and performed one round of
screening on the mixed pool against IgG. The result showed that 20 fold higher
amount of BDA molecules were selected out of mixed pool than which of PDO
molecules.

The validity of cDNA display has
been proved though, some research still needed to be done to improve this
method. In order to resolve the problem that the productivity of
cDNA-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 regulated
some conditions like reducing buffer exchange for His-tag purification and
increasing the amount of SA beads 39. Recently an optimized puromycin linker
40 and a photo-cross-linker 41 for cDNA display were reported. In terms of
in vitro selection speed, by integrating transcription and translation into on
step and skipping the ligation between mRNA and puromycin-linker, 6 rounds of
selections can be performed within 14 h, making the display selection less time-consuming
42.

1.4  IVC (in vitro compartmentalization)

Although different from natural compartments, like bacteria
and yeast, artificial compartments can also serve the purpose of coupling gene
and its encoded protein in separated space. Water-in-oil (W/O) emulsion
droplets have been used as such man-made compartments to allow for
transcription and translation of individual gene proceed separately 43.

Normally the water-in-oil emulsion is prepared by stirring an
aqueous solution containing a library of genes and in vitro expression system
into an oil-surfactant mixture. After transcription and translation, genes can
be associated with gene products through covalent linkage or microbeads. Then
the emulsion is broken and selection is performed. Finally, the target gene is
enriched through PCR.

IVC has two advantages in terms of enzyme evolution. Except
being capable of processing large library size (108–1011
genes), IVC can select for more enzyme properties, such as regulatory and
catalytic activity, than just binding activity. Moreover, it allows for
selection of enzyme with multiple turnover. There are still some limitations
exist during screening which researchers have been working on. Griffths et al
first combined water-in-oil-in-water double emulsion and FACS system together
for directed evolution of b-galactosidase and got a product sorting rate of
20000 droplets s-144. However, some limitations, like the
polydispersity of droplets, were noticed. To get rid of those limitations they
developed a droplet-based microfluidic system instead of FACS, which resulted
in an order of magnitude lower polydispersity than FACS system at a cost of
10-fold lower screening speed45. Some other study also applied microfluidic
platform, for example screening for activity of FeFe hydrogenase46, hydrolytic
activities of a promiscuous sulfatase47 and glucose oxidase activity. On the
other hand, other studies focused on some improvements when using FACS system
for screening, like a generalizable protocol of producing monodisperse
picolitre double emulsion droplets for directed evolution48 and a protocol employing
membrane-extrusion technique to generate uniform emulsion droplets49.

 JD1?

Needs rephrasing, not clear.

 JD2?????

 JD3??
Don’t understand this.

 JD4?
Is this an advantage?

 JD5How
does translation compare with gene randomization?

 JD6Review?

 JD7What
you describe under this title are different genotype-phenotype linkages
(display methods) which are used for in vitro directed evolution. The title is
much broader than the contents and needs revision. Display is just one part of
the directed evolution process.

 JD8?

 JD9What
property?

 JD10What
does this mean?

 JD11Does
this mean the previous one is not?

x

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