An efficient cell-free protein synthesis platform for producing proteins with pyrrolysine-based noncanonical amino acids.

Incorporation of noncanonical amino acids (ncAAs) into proteins opens new opportunities in biotechnology and synthetic biology. Pyrrolysine (Pyl)-based ncAAs are some of the most predominantly used, but expression systems suffer from low yields. Here, we report a highly efficient cell-free protein synthesis (CFPS) platform for site-specific incorporation of Pyl-based ncAAs into proteins using amber suppression. This platform is based on cellular extracts derived from genomically recoded Escherichia coli lacking release factor 1 and enhanced through deletion of endonuclease A. To enable ncAA incorporation, orthogonal translation system (OTS) components (i.e., the orthogonal transfer RNA [tRNA] and orthogonal aminoacyl tRNA synthetase) were coexpressed in the source strain prior to lysis and the orthogonal tRNACUA Pyl that decodes the amber codon was further enriched in the CFPS reaction via co-synthesis with the product. Using this platform, we demonstrate production of up to 442 ± 23 µg/mL modified superfolder green fluorescent protein (sfGFP) containing a single Pyl-based ncAA at high (>95%) suppression efficiency, as well as sfGFP variants harboring multiple, identical ncAAs. Our CFPS platform can be used for the synthesis of modified proteins containing multiple precisely positioned, genetically encoded Pyl-based ncAAs. We anticipate that it will facilitate more general use of CFPS in synthetic biology.

Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.