Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E. coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2-anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.
"The presence of release factor 1 (RF1) can cause early termination of proteins when using amber suppression technology (Johnson et al., 2011; Hong et al., 2014). Recent advances have addressed some of these challenges by improving NSAA incorporation efficiency by engineering o-tRNA (Young et al., 2010; Chatterjee et al., 2012), o-aaRS (Liu et al., 1997; Chatterjee et al., 2012), or EF-Tu (Doi et al., 2007; Park et al., 2011) as well as controlling transcription and translation rate (Young et al., 2010; Chatterjee et al., 2013), and removing RF1 competition (Mukai et al., 2010; Johnson et al., 2011; Loscha et al., 2012; Lajoie et al., 2013). While further efforts to re-engineer translation are still needed, these improvements are accelerating rapid growth in synthetic biology efforts to “upgrade protein synthesis” (O'Donoghue et al., 2013). "
[Show abstract][Hide abstract] ABSTRACT: Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.
Frontiers in Chemistry 06/2014; 2:34. DOI:10.3389/fchem.2014.00034
"Plasmid vector for in vitro translation was constructed by transferring the coding sequence of the engineered Renilla Luciferase  to the pGSH vector, which was kindly provided from Prof. Ohtsuki . The engineered Renilla Luciferase was translated using in vitro translation system (TnT Coupled Reticulocyte Lysate System; Promega). "
[Show abstract][Hide abstract] ABSTRACT: RNA and protein are potential molecules that can be used to construct functional nanobiomaterials. Recent findings on riboswitches emphasize on the dominative function of RNAs in regulating protein functions through allosteric interactions between RNA and protein. In this study, we demonstrate a simple strategy to obtain RNAs that have a switching ability with respect to protein function in response to specific target molecules. RNA aptamers specific for small ligands and a trans-activation-responsive (TAR)-RNA were connected by random RNA sequences. RNAs that were allosterically bound to a trans-activator of transcription (Tat)-peptide in response to ligands were selected by repeated negative and positive selection in the absence and presence of the ligands, respectively. The selected RNAs interacted with artificially engineered Renilla Luciferase, in which the Tat-peptide was inserted within the Luciferase, in the presence of the specific ligand and triggered the "Lighting-UP" switch of the engineered Luciferase.
PLoS ONE 03/2013; 8(3):e60222. DOI:10.1371/journal.pone.0060222 · 3.23 Impact Factor
"We believe that more precise experiments will be required to determine the compatibility of D- and β-amino acids with ribosomal translation systems. Following this reevaluation of nonproteinogenic amino acids, further studies on the engineering of translation factors, such as the ribosome [134–136], elongation factor Tu [137, 138], and tRNA [139–141], will be required to overcome these limitations. The second problem is the low cell permeability of peptides. "
[Show abstract][Hide abstract] ABSTRACT: The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.
Journal of nucleic acids 10/2012; 2012:713510. DOI:10.1155/2012/713510
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