Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

Abstract

Cell-free protein synthesis (CFPS) has emerged as a novel protein expression platform. Especially the incorporation of non-canonical amino acids (ncAAs) has led to the development of numerous flexible methods for efficient and extensive expression of artificial proteins. Approaches were developed to eliminate the endogenous competition for ncAAs and engineer translation factors, which significantly enhanced the incorporation efficiency. Furthermore, in vitro aminoacylation methods can be conveniently combined with cell-free systems, extensively expanding the available ncAAs with novel and unique moieties. In this review, we summarize the recent progresses on the efficient and extensive incorporation of ncAAs by different strategies based on the elimination of competition by endogenous factors, translation factors engineering and extensive incorporation of novel ncAAs coupled with in vitro aminoacylation methods in CFPS. We also aim to offer new ideas to researchers working on ncAA incorporation techniques in CFPS and applications in various emerging fields.

Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming

“…For challenging ncAAs, competing processes can be significant hurdles for efficient incorporation. , Two of thesenear-cognate misincorporation and peptidyl-tRNA dropoffhave been studied in vivo under conditions of amino acid starvation, − but these processes are likely to also manifest themselves with difficult to translate ncAAs in vitro. A careful inspection of the literature shows that the majority of the studies with difficult ncAAs utilize reconstituted in vitro translation reactions that are devoid of AA-tRNAs that are not required for the encoding mRNA (presumably to minimize near-cognate misincorporation) and have a C-terminal tag to remove sequences that result from misincorporations within the purification-tag region or peptidyl-tRNA dropoff. , While these strategies do lead to homogeneous peptides, they hide the underlying inefficiencies that compromise yield and fidelity.…”

Chemically Acylated tRNAs are Functional in Zebrafish Embryos

“…In fact, chemically acylated tRNAs and their use for UAA incorporation into proteins in vitro was first reported in 1989 by the Schultz group . To conduct these experiments in cells, delivery of the acylated tRNA represents an obstacle that was solved through injection into large frog oocytes. , …”

“…11 To conduct these experiments in cells, delivery of the acylated tRNA represents an obstacle that was solved through injection into large frog oocytes. 12,13 Surprisingly, the utilized oocytes have always been unfertilized, limiting the complexity of the system and of the biological studies. Here, we demonstrate for the first time that injection of chemically acylated tRNA into fertilized zebrafish oocytes results in robust UAA incorporation into proteins during embryo development that is comparable to enzymatic genetic code expansion in this model organism.…”

A CHO-Based Cell-Free Dual Fluorescence Reporter System for the Straightforward Assessment of Amber Suppression and scFv Functionality

“…Furthermore, CFPS can be performed using linear templates in high-throughput microliter scale (Dondapati et al, 2020;Silverman et al, 2020). By simply adding an arbitrary orthogonal tRNA/tRNA aminoacyl-synthetase pair, a compatible ncAA with a reactive moiety, and a template with an amber stop codon in its coding sequence to the cell-free reaction, active ncAA-proteins can be produced ready to be conjugated site-specifically to a matching reactive compound without prior purification (Gao et al, 2019;Wu et al, 2020;Stech et al, 2021). Although antibodies are commonly produced in CHO cells (Dalton and Barton, 2014) with in vivo production in medium and large scale being more economic compared to CHO-based CFPS (Thaore et al, 2020), we deem practical and economic advantages in using CFPS for initial screening of tRNA/aaRS/ncAA triplets and small scale production for characterization of ncAA-containing proteins.…”