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

Abstract

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.

Fig. 1

CFPS from extracts of a genomically recoded organism. a Schematic of the production and utilization of crude extract from genomically recoded organisms with plasmid overexpression of orthogonal translation components for cell-free protein synthesis (CFPS). CFPS reactions are supplemented with the necessary substrates (e.g., amino acids, NTPs, etc.) required for in vitro transcription and translation as well as purified orthogonal translation system (OTS) components to help increase the ncAA incorporation efficiency. aaRS, aminoacyl tRNA synthetase; ncAA, non-canonical amino acid; T7P, T7 RNA polymerase; UAG, amber codon. b Time course of superfolder green fluorescent protein (sfGFP) synthesis catalyzed by extracts derived from a genomically recoded organism, C321.∆A, and a commercial strain, BL21 Star (DE3). Three independent batch CFPS reactions (n = 3) were performed at 30 °C for each time point over 24 h. Error bar = 1 SD

Fig. 2

Engineering C321.∆A variants for enhanced CFPS. a Schematic of design-build-test cycles employing multiplex automated genome engineering (MAGE) to disrupt putative negative protein effectors (Supplementary Table 1) in engineered C321.∆A strains for producing extracts with enhanced cell-free protein synthesis (CFPS) yields. b Cell extracts derived from C321.∆A and genomically engineered strains containing a single putative negative effector inactivation were screened for sfGFP yields. Beneficial mutations that increase active yields ≥ 50% relative to C321.ΔA are highlighted with an *(p < 0.01, Student’s t-test). c C321.∆A.542 (endA⁻) was chosen as the next base strain and the following beneficial disruptions were pursued in combination: rne, mazF, tnaA, glpK, lon, and gor. d C321.∆A.709 (endA⁻ gor⁻) was selected as the subsequent base strain for triple and quadruple mutant construction. C321.∆A.759 (endA⁻ gor⁻ rne⁻ mazF⁻) yielded the highest level of CFPS production. Total sfGFP concentration was measured by counting radioactive ¹⁴C-Leucine incorporation and active protein was measured using fluorescence. Three independent batch CFPS reactions were performed for each sample at 30 °C for 20 h (n = 3). Error bar = 1 SD

Fig. 3

Multi-site incorporation of pAcF into proteins. Cell-free p-acetyl-l-phenylalanine (pAcF) incorporation was compared using extracts derived from BL21 Star (DE3), MCJ.559, and C321.∆A.759 strains containing the pEVOL-pAcF vector. The pEVOL-pAcF vector harbors the orthogonal translation machinery necessary for pAcF incorporation. a Total protein yields for wild-type (WT) and 1 UAG versions of superfolder green fluorescent protein (sfGFP) and chloramphenicol acetyl transferase (CAT) are shown along with an autoradiogram of the resulting protein product. Supplementary Fig. 8 shows the entirety of the autoradiogram along with a molecular weight marker. b Multi-site incorporation of pAcF into sfGFP variants as quantified by active protein produced. The sfGFP variants used were wild-type (WT), sfGFP containing a single pAcF corresponding to the position of T216 (1 UAG), sfGFP containing sfGFP containing two pAcFs (2 UAG), and sfGFP containing five pAcFs (5 UAG). Three independent batch CFPS reactions were performed for each sample at 30 °C for 20 h (n = 3). Error bar = 1 SD. c Spectrum of the 28 + charge state of sfGFP, obtained by top-down mass spectrometry and illustrating site-specific incorporation of pAcF at single and multiple sites. Experimental (Exper) and theoretical (Theor) mass peaks for each sfGFP variant are shown. Major peaks (color) in each spectrum coincide with the theoretical peaks for each species (see also Supplementary Fig. 11). Smaller peaks immediately to the right of the major peaks are due to oxidation of the protein, a common electrochemical reaction occurring during electrospray ionization. Experimentally determined masses are ≤ 1 p.p.m. in comparison of theoretical mass calculations. Owing to the size of pAcF, misincorporation would result in peaks present at lower m/z values relative to the colored theoretical peak

Fig. 4

Multi-site ncAA incorporation at high yield and purity. a Schematic of the protein sequences for wild-type ELPs containing three pentapeptide repeats per monomer unit (ELP-WT) and ELPs containing 1 ncAA per monomer unit (ELP-UAG). b SDS-PAGE and autoradiogram analysis of cell-free produced ELP-WT and ELP-UAG 20-, 30-, and 40-mers in the presence ( + ) and absence ( – ) of p-acetyl-L-phenylalanine (pAcF). Numbers next to the molecular weight ladder (L) represent the approximate kilodalton (kDa) size of the band. c Total protein yields of cell-free synthesized ELPs (20-, 30, and 40-mers) after incubation at 30 °C for 20 h are shown. Three independent batch CFPS reactions were performed for each sample (n = 3). Error bar = 1 SD. d–f Deconvoluted mass spectra of ELPs obtained by top-down mass spectrometry illustrate complete, site-specific incorporation of pAcF at d 20, e 30, and f 40 sites. Deconvoluted average masses for the major peaks in each spectrum (Exper) match the theoretical average mass (Theor) for each species within 1.2 Da. Smaller peaks next to the major peaks arise from minor oxidation of the protein during electrospray ionization