pet28 sfgfp 150tag  (Addgene inc)

Journal: Journal of molecular biology

Article Title: Overcoming near-cognate suppression in a Release Factor 1-deficient host with an improved nitro-tyrosine tRNA synthetase

doi: 10.1016/j.jmb.2020.06.014

Figure Lengend Snippet: Whole protein mass spectrometry of purified, nitrated sfGFP expressed using the 2nd-generation 5B aaRS in a +RF1 and a −RF1 expression host shows mis-incorporation of natural amino acids in the - RF1 system. (A) Wild-type sfGFP, (B) sfGFP-150TAG protein expressed in RF1-deficient (B-95) cells without any GCE machinery contains peaks consistent with insertion of Gln, Glu or Lys at site 150 (although we cannot distinguish between Q, E and K, for clarity we denote these peaks as simply Q), (C) sfGFP-150-nitroTyr (nY150) expressed in +RF1 strain, (D) 150-nitroTyr (nY150) expressed in −RF1 strain (E) sfGFP-134/150-nitroTyr (nY134/nY150) expressed in +RF1 strain and (F) sfGFP-134/150-nitroTyr (nY134/nY150) expressed in −RF1 strain, which contains peaks consistent with one (nY/Q) or two (Q134/Q150) Gln, Glu or Lys residues inserted. -Met: loss of N-terminal methionine. Measured and expected mass are omitted for clarity and listed in Supplemental Table S1. Purple asterisks are peaks with masses consistent with Na+/K+ adducts.

Article Snippet: Chemically competent BL21-ai or B-95(DE3) Δ A Δ fabR (referred to B-95) were co-transformed with pET28-sfGFP-150TAG (containing a C-terminal His 6 affinity purification tag, Addgene #85493) and the appropriate GCE machinery plasmid.

Techniques: Mass Spectrometry, Purification, Expressing

Journal: Journal of molecular biology

Article Title: Overcoming near-cognate suppression in a Release Factor 1-deficient host with an improved nitro-tyrosine tRNA synthetase

doi: 10.1016/j.jmb.2020.06.014

Figure Lengend Snippet: Analysis of nitroTyr binding on the active site of the “5B” aaRS modified by single residue alterations. (A) Crystal structure of the “5B” nitroTyr aaRS active site highlighting the ten residues analyzed for mutational analysis by Rosetta (PDB 4nda). (B) Analysis of shape complementarity (top) and ligand binding interface energy (bottom) for nitroTyr to each modified residue (numbers are listed on top of each panel). Black outlined boxes indicate wild-type amino acids. Value for each amino acid position is subtracted from the original value to show the absolute effect of each mutation. In both plots, blue means improved over original value. Values are provided in the Supplemental Dataset. (C) Variants of “5B” aaRS C70A, S158C and C70A/S158C predicted to have better activity were tested for their ability to express sfGFP-150TAG in vivo in RF1-containing BL21 cells by measuring in-cell fluorescence normalized to culture density. Cultures were supplemented with 0, 0.1 and 1 mM nitroTyr as indicated. Error bars represent the standard deviation of three independent cultures.

Article Snippet: Chemically competent BL21-ai or B-95(DE3) Δ A Δ fabR (referred to B-95) were co-transformed with pET28-sfGFP-150TAG (containing a C-terminal His 6 affinity purification tag, Addgene #85493) and the appropriate GCE machinery plasmid.

Techniques: Binding Assay, Modification, Ligand Binding Assay, Mutagenesis, Activity Assay, In Vivo, Fluorescence, Standard Deviation

Journal: Journal of molecular biology

Article Title: Overcoming near-cognate suppression in a Release Factor 1-deficient host with an improved nitro-tyrosine tRNA synthetase

doi: 10.1016/j.jmb.2020.06.014

Figure Lengend Snippet: Evaluation of a complete nitroTyr aaRS library at sites 70 and 158 of the “5B” nitroTyr aaRS. (A) The pool of 400 unique protein variants at sites 70 and 158 were selected for their ability to incorporate nitroTyr and no canonical amino acids and surviving members were evaluated for their ability to incorporated nitroTyr into sfGFP-150TAG (see Materials and Methods). Sequences for the top 16 performing hits are indicated, revealing seven unique variants. (B) Rosetta-predicted shape complementarity and ligand interface energy of all 400 variants (left panel), and expanded view of 80 variants with shape complementarity > 0.6 and ligand interface energy < −10 (right panel). The seven new experimental hits (colored circles) and the four previously characterized aaRSs (colored diamonds) were all scored by Rosetta as relatively favorably by these parameters. The top five variants in each category (ligand interface energy and shape complementarity) are listed in Supplemental Table S4.

Article Snippet: Chemically competent BL21-ai or B-95(DE3) Δ A Δ fabR (referred to B-95) were co-transformed with pET28-sfGFP-150TAG (containing a C-terminal His 6 affinity purification tag, Addgene #85493) and the appropriate GCE machinery plasmid.

Techniques:

Journal: Journal of molecular biology

Article Title: Overcoming near-cognate suppression in a Release Factor 1-deficient host with an improved nitro-tyrosine tRNA synthetase

doi: 10.1016/j.jmb.2020.06.014

Figure Lengend Snippet: Functional characterization of the “A7” nitroTyr aaRS. (A) In-cell fluorescence normalized to culture density of cells expressing sfGFP-150TAG and sfGFP-134/150TAG using either the “5B” or “A7” aaRS in the presence of either 0, 0.1 and 1 mM nitroTyr in the media. All expressions were performed in RF1-containing, BL21 cells (see Materials and Methods for details). (B) Normalized fluorescence of RF1-containing BL21 cells expressing sfGFP-150TAG measured as a function of nitroTyr supplemented to the media allows for evaluation of the UP50, the concentration at which half maximal fluorescence is achieved. Curves are fitted to a standard Michaelis-Menton like model. (C) Comparison of sfGFP-134/150TAG expression in defined vs. complex (ZY-based) auto-induction media using the “A7” and “5B” nitroTyr aaRSs with RF-1 containing BL21 cells (see Materials and Methods). The dual TAG reporter of sfGFP was used to emphasize efficacy of the “A7” aaRS in complex media compared to the “5B”. (D) Whole protein mass spectra of sfGFP (gray, expected/observed: 27827/27826 Da), sfGFP-150TAG (green, expected/observed: 27921/27919 Da) and sfGFP 134/150TAG (blue, expected/observed: 28013/28012 Da) expressed using the “A7” nitroTyr aaRS in RF1-deficient B-95 cells shows homogenous incorporation of nitroTyr and no detectable natural amino acids. For panels A-C, error bars represent standard deviations from three independent cultures.

Article Snippet: Chemically competent BL21-ai or B-95(DE3) Δ A Δ fabR (referred to B-95) were co-transformed with pET28-sfGFP-150TAG (containing a C-terminal His 6 affinity purification tag, Addgene #85493) and the appropriate GCE machinery plasmid.

Techniques: Functional Assay, Fluorescence, Expressing, Concentration Assay