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.