Introduction
Recombinant in vitro transcription-translation (TX-TL)
procedures, also known as cell-free systems, have becoming increasingly
common in recent years for a variety of biochemical and molecular
procedures, mainly due to their versatility, and particularly in
synthetic biology (Hammerling, Krüger, & Jewett, 2019; Keasling, 2012;
Perez, Stark, & Jewett, 2016; Villarreal & Tan, 2017). These systems
allow flexibility in the reaction condition of parameters for protein
production, such as the transcription and translation machinery, as well
as minimizing the possible limitations that carry working with living
cells (Carlson, Gan, Hodgman, & Jewett, 2012; Hodgman & Jewett, 2012;
Silverman, Karim, & Jewett, 2019). Two main types of cell-free systems
exist: those that derive from cell extracts and those that use purified
recombinant proteins. The cell lysate-based systems were the first to be
developed, and employ extracts from a variety of living organisms such
as, Escherichia coli , yeast, fall armyworm, wheat germ, tobacco,
rabbit reticulocytes and HeLa cell line (Anderson, Straus, & Dudock,
1983; Buntru, Vogel, Spiegel, & Schillberg, 2014; Ezure, Suzuki, &
Ando, 2014; Jackson & Hunt, 1983; Sun et al., 2013; Wang, Zhao, &
Zhao, 2014; Yadavalli & Sam-Yellowe, 2015). The application of using
cell lysates quickly gained popularity as it tackled constraints
encountered when performing synthetic biology research using living
organisms, such as expression of protein that exhibits cellular toxicity
or protein production under growth toxic compound (Bowie et al., 2020;
Kay & Jewett, 2020; Tinafar, Jaenes, & Pardee, 2019). These systems
often produce high yield of protein products; however, some issues still
remain due to components that actively degrade the mRNA and proteins
(nucleases, proteases), and the presence of additional unknown factors
included in the cell lysate. While ongoing research continues to
mitigate these issues (Didovyk, Tonooka, Tsimring, & Hasty, 2017;
Fujiwara & Doi, 2016), cell-free systems using recombinant protein
elements and purified ribosomes such as PURE system (Kuruma & Ueda,
2015; Y Shimizu et al., 2001; Yoshihiro Shimizu, Kuruma, Kanamori, &
Ueda, 2014), offer a contaminant-free alternative with a final
significant protein yield (Kazuta, Matsuura, Ichihashi, & Yomo, 2014).
Lately, more research has focused on simplification, robustness and
low-cost for reconstruction (Lavickova & Maerkl, 2019) as well as
altering the energy source (Wang et al., 2019) in the PURE system.
These advantages of recombinant systems are particularly beneficial when
one needs a control over conditions for high-throughput screening and
directed evolution of peptide/proteins (Contreras-Llano & Tan, 2018;
Dodevski, Markou, & Sarkar, 2015; Fujii et al., 2014). Different
display methods (phage display (Ledsgaard, Kilstrup, Karatt-Vellatt,
McCafferty, & Laustsen, 2018), yeast display (Boder & Wittrup, 1997;
Cherf & Cochran, 2015), ribosome display (Zahnd, Amstutz, & Plückthun,
2007), liposome display (Fujii, Matsuura, Sunami, Kazuta, & Yomo,
2013), DNA display (Bertschinger & Neri, 2004; Doi & Yanagawa, 1999;
Yonezawa, Doi, Kawahashi, Higashinakagawa, & Yanagawa, 2003), cDNA
display (Naimuddin & Kubo, 2016; Yamaguchi et al., 2009), mRNA display
(Nemoto, Miyamoto-Sato, Husimi, & Yanagawa, 1997; Roberts & Szostak,
1997; Seelig, 2011)) use various strategies to couple genotype to
phenotype and as such have become indispensable tools for directed
evolution. Among the display methods, in vitro approaches such as
mRNA, cDNA, and ribosome display can screen the highest number of
molecules (up to 1013) to be tested because they are
not limited by the efficiency of transformation or transfection. In the
case of mRNA display, screening of large libraries is achieved by
creating a covalent phenotype-genotype linkage between an mRNA and the
polypeptide it encodes using puromycin (Takahashi, Austin, & Roberts,
2003). Moreover, in vitro reactions can be easily modified to
suit a specific environment for functional screening (Josephson,
Ricardo, & Szostak, 2014).
The utility of mRNA display is limited by the relative instability of
mRNA-protein conjugates, especially in cell lysate-based translation
systems, due to the presence of proteases and especially ribonucleases
(Hino et al., 2008; Opyrchal, Anderson, Sokoloski, Wilusz, & Wilusz,
2005; Shin & Noireaux, 2010). The use of RNase inhibitors and
nuclease-free chemicals can help minimize the degradation of RNA
components (Newton, Cabezas-Perusse, Tong, & Seelig, 2020; Seelig,
2011). The recent advent of reconstituted contaminant-free PURE
translation system has made in vitro display methods more popular
for screening antibodies (Kanamori, Fujino, & Ueda, 2014; Nagumo,
Fujiwara, Horisawa, Yanagawa, & Doi, 2016) and functional
peptidomimetics (Bashiruddin & Suga, 2015). Since the PURE system
operates primarily with reconstituted components, it offers increased
stability of mRNA-protein conjugates. In addition, the cDNA display
method, which converts translated mRNA-peptide conjugates into
mRNA/cDNA-peptide conjugates, is advantageous under conditions where RNA
instability is an issue during the selection step, such as targeting
cell surface antigens under the presence of cellular ribonucleases (Ueno
& Nemoto, 2012; Yamaguchi et al., 2009).
Here, we have established robust PURE system-based mRNA display and cDNA
display methods, and compare their performance to screen for FLAG
epitope sequences against anti-FLAG M2 antibody (Roosild, Castronovo, &
Choe, 2006). Next-generation sequencing has been recently used along
with display technology to provide an overview of sequence distribution
(Fujimori et al., 2012). Hence, we performed round-by-round deep
sequencing to validate our method by monitoring stepwise enrichment
patterns of the core FLAG epitope motifs and contribution of key
residues that would otherwise be difficult to trace by traditional
sequencing methods.