Introduction can be distinguished based on the use of

Introduction

      Directed
evolution technologies of proteins have been developed many decades for the
purpose of desired properties or other specific characteristics. Different
methods of library analysis have been found to identify the desired variants,
and there are two broad categories of methods: screening and selection. Among
selection methods, display technologies are widely used as a high-throughput
selection tool since their expressed protein can easily access to the external
environment, 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 membranes, and relatively 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 diversity 5.

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This review will introduce the mechanism as well as the
methodology of mRNA display and ribosome display, which are the two frequently
used in vitro directed evolution
technologies, 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 technologies

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
selection is illustrated (Figure 1).

Formed
mRNA-ribosome-protein ternary complex is used for affinity selection. After
elution, the mRNA of enriched protein 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. First, the template
should comprise of T7 promoter, 5′-stemloop, ribosome binding site, regulatory
sequence for translation, DNA fragment and spacer containing a region of 3′-stemloop
is necessary (Figure 2).

The
presence of stemloops is important for resistance to RNase which act 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 need to be added to prevent the
rescue of stalled ribosome 28. Third, in the case of very few mRNA is
recovered 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 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 its 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 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 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 physical 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 tRNA. At the end of the transcription, attached puromycin enters the A site
of ribosome and transferred to the growing peptide chain, in turn stalls the
ribosome and forms a covalent bond between mRNA and corresponding peptide. The
typical scheme of a single round of mRNA display selection is showed (Figure
4).

Briefly, a given DNA library is converted to the mRNA library
through transcription, which is 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.