Robust production of recombinant phosphoproteins using cell-free protein synthesis

Abstract

Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here, we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signaling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly-phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure-function relationships, kinase signaling networks, and kinase inhibitor drugs.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

“…The amino acid compositions were listed in Table 1 (MS spectra were listed in Fig.S2-S10). When the total tRNA was added, tyrosine, glutamine, lysine, and glycine were detected which is consistent with the previous report [22]. Additionally, we detected serine residue at position 151 in the PURE reaction which may result from the dephosphorylation of Sep. With ∆QYK tRNAs added, we did not detect tyrosine, glutamine, and lysine residues at position 151, and the MS quality was improved for Sep incorporation (Figure S7 and S8).…”

“…Previous studies showed that the contamination of canonical amino acids at amber codons in CFPS systems is mainly from glutamine, as well as low level of tyrosine, glutamate, lysine, and glycine [22, 24], which is reasonable because codons for tyrosine (UAU and UAC), lysine (AAG), glutamine (CAG) and glutamate (GAG) have only one nucleotide difference from amber codon (UAG). In E. coli , there is only one species of tRNA isoacceptor for glutamate and lysine, respectively, while tyrosine and glutamine have two species of tRNA isoacceptors, individually [31].…”

“…We chose superfolder green fluorescent protein (sfGFP) as a reporter, which was engineered to have improved tolerance of circular permutation, greater resistance to chemical denaturants, and enhanced folding kinetics, thus being widely used in the field of genetic code expansion [32, 33]. And we selected phosphoserine (Sep) incorporation system [29], which recently has been used in CFPS systems to make phosphoprotein but with the contamination from near-cognate suppression [22]. …”

“…Most notably, the first genomically recoded E. coli strain was developed, in which all 321 TAG codons were mutated to synonymous TAA codons, allowing the deletion of RF1 without observed growth defects [21]. A similar strategy was also applied in CFPS systems with significantly improved ncAA incorporation, especially for the incorporation of identical ncAAs at multiple positions in target proteins [22-25]. Nevertheless, it has been noticed that natural suppression of amber codon by near cognate tRNAs exists under the circumstance of RF1 deletion, which results in impurity of amino acid composition at designed positions with amber codons [17, 21, 26, 27].…”

Translation system engineering in Escherichia coli enhances non‐canonical amino acid incorporation into proteins

“…For example, photocaged (Wu et al, 2004), fluorescent (Summerer et al, 2006), and bio-orthogonal reactive (Chin et al, 2002a,b; Lang et al, 2012) ncAAs have provided new ways to study protein structure and dynamics (Liu and Schultz, 2010). In addition, ncAAs that mimic natural post-translational modifications have helped to elucidate the role of such modifications in previously unattainable ways (Bröcker et al, 2014; Davis and Chin, 2012; Lee et al, 2013; Neumann et al, 2009; Oza et al, 2015; Park et al, 2011; Tian et al, 2014). Further, ncAA incorporation into proteins has opened the way to novel antibody drug conjugates (Axup et al, 2012; Zimmerman et al, 2014), modified human therapeutics (Cho et al, 2011), and protein biomaterials (Albayrak and Swartz, 2014), among other applications.…”