Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

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.

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

“…Cell-free protein synthesis (CFPS) is gaining ground as a method for ncAA incorporation, offering several advantages [7, 8]. First, CFPS can benefit from the relatively slower synthesis and the greater distance between ribosomes for proper protein folding [9].…”

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…169,170 The ability to incorporate unnatural aminoacids into a given protein in a site-specific manner opens a wide array of opportunities, including easy incorporation of biomarkers, fluorescent or radio-labels for both research and diagnostic applications, and access to new categories of sequence-defined biopolymers. 171 Some of these applications are more directly applicable to vaccines, 172 including site-specific conjugation of polysaccharide structures, addition of immunogenic amino acids to break tolerance and generate vaccines targeted against autologous proteins associated with cancer, the ability to achieve modifications such as lipidation which are otherwise difficult to carry out in standard conditions, and the ability to mimic eukaryotic post-translational modifications in prokaryotic bacterial hosts. Of note, non-natural amino acids could play a role as adjuvants, knowing that, for example, lipidation is a key factor of the immunogenicity of some vaccine antigens.…”

Preparation of Amino Acid Mixtures for Cell-Free Expression Systems

“…This method represents an economical and easy way to solubilize amino acids for in vitro protein synthesis and could be an alternative to the widely used, yet expensive commercial kits. Although only tested for an all E. coli cell-free system, we expect that our protocol could be adapted for other cell-free expression systems, such as T7-based E. coli systems; wheat germ, yeast, plant, and mammalian crude extracts; or platforms developed for the incorporation of nonstandard amino acids (15). In addition, the flexibility of our method may allow for combinatorial studies involving machine learning and high-throughput experimentation (16).…”