Abstract
More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.
Highlights
Twenty amino acids composing huge repertoire of proteins with the remarkably diverse structure and function are encoded by the universal genetic code of all known organisms [1] with the rare exceptions of selenocysteine being incorporated in response to the stop codon UGA [2,3] and pyrrolysine specified by the UAG codon [3,4]
Many diverse techniques were developed for the incorporation of noncanonical amino acids (NAAs) into proteins to introduce new functional groups apart from those found in the canonical amino acids including chemical approach based on direct modification or synthesis of the desired proteins [9,10,11], and biosynthetic methods to totally replace canonical amino acid to their close structural analogues in auxotrophic bacteria [12,13,14,15] or co-translationally incorporate NAAs into target proteins by utilizing chemically modified aminoacylated tRNA molecules for in vitro translation [16,17,18,19] or for Xenopus oocytes [20,21,22]. These approaches for the generation of NAA-incorporated proteins are extremely useful for altering protein structure and properties and can be applied for a large variety of NAAs, their application is limited by the selectivity and overall efficiency of the methodology
An ideal methodology to incorporate NAAs into recombinant proteins was considered to exploit the translational machinery of the host cell in the same manner as the canonical amino acids, enables specific changes to be precisely made in proteins directly in vivo, providing novel tools for understanding biology in molecular terms in the native settings
Summary
Twenty amino acids composing huge repertoire of proteins with the remarkably diverse structure and function are encoded by the universal genetic code of all known organisms [1] with the rare exceptions of selenocysteine being incorporated in response to the stop codon UGA [2,3] and pyrrolysine specified by the UAG codon [3,4]. Many diverse techniques were developed for the incorporation of NAAs into proteins to introduce new functional groups apart from those found in the canonical amino acids including chemical approach based on direct modification or synthesis of the desired proteins [9,10,11], and biosynthetic methods to totally replace canonical amino acid to their close structural analogues in auxotrophic bacteria [12,13,14,15] or co-translationally incorporate NAAs into target proteins by utilizing chemically modified aminoacylated tRNA molecules for in vitro translation [16,17,18,19] or for Xenopus oocytes [20,21,22] These approaches for the generation of NAA-incorporated proteins are extremely useful for altering protein structure and properties and can be applied for a large variety of NAAs, their application is limited by the selectivity and overall efficiency of the methodology. We are focused on the description of the methodology and the recent progress in the field of E. coli genetic code expansion with the emphasis on the nonsense suppression, since to date, it is the most robust, established and demanded techniques for the recombinant protein production
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