purexpress in vitro transcription translation system  (New England Biolabs)

Journal: bioRxiv

Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

doi: 10.1101/653360

Figure Lengend Snippet: Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

Article Snippet: The PURExpress in vitro transcription-translation system was purchased from NEB.

Techniques: Construct, Software

Journal: bioRxiv

Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

doi: 10.1101/653360

Figure Lengend Snippet: Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

Article Snippet: The PURExpress in vitro transcription-translation system was purchased from NEB.

Techniques: Pulse Chase, Incubation, Construct

Journal: Biology Open

Article Title: Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

doi: 10.1242/bio.032862

Figure Lengend Snippet: Reduced tyrosine autophosphorylation of in vitro -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression system. Coupled in vitro transcription-translation reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P

Article Snippet: A reconstituted E. coli -based in vitro transcription-translation system (PURExpress, New England Biolabs, Beverley, MA, USA) was used to express a DYRK1A(28-499) construct fused to an N-terminal Strep-tag 2 sequence.

Techniques: In Vitro, Construct, Derivative Assay, Expressing, Incubation, Recombinant

Journal: Biology Open

Article Title: Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

doi: 10.1242/bio.032862

Figure Lengend Snippet: Reduced tyrosine autophosphorylation of in vitro -translated DYRK1A-L295F. DYRK1A constructs (28-499) were expressed in a cell-free E. coli- derived expression system. Coupled in vitro transcription-translation reactions were incubated for 90 min at 37°C before reaction products were subjected to immunoblot analysis. For quantification, pTyr signals were normalized to the total amounts of recombinant DYRK1A as determined by detection of the N-terminal StrepTag (means and s.d., n =3). The difference in relative tyrosine autophosphorylation was tested for statistical significance by one-sample t -test (* P

Article Snippet: In vitro translation A reconstituted E. coli -based in vitro transcription-translation system (PURExpress, New England Biolabs, Beverley, MA, USA) was used to express a DYRK1A(28-499) construct fused to an N-terminal Strep-tag 2 sequence.

Techniques: In Vitro, Construct, Derivative Assay, Expressing, Incubation, Recombinant

Journal: Nucleic Acids Research

Article Title: Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA

doi: 10.1093/nar/gkab608

Figure Lengend Snippet: Tre23 inhibits protein synthesis. ( A ) Phase contrast and fluorescence microscopy recordings of E. coli cells constitutively producing short-lived green fluorescent protein (GFP-LVA) and the Tre23 toxin from the low-copy number pNDM220 vector, before and 100 min post toxin induction. Cells bearing the empty pNDM220 vector (Ø) were used as control. ( B ) Replication, transcription and translation rates before (time 0) and 60 min after the induction of Tre23 toxin from pNDM220 vector (red line) as compared to control carrying empty vector (black line) were measured by the incorporation (in count per minute, cpm) of [ 3 H] thymidine, [ 3 H] uridine and [ 35 S] methionine, respectively. ( C ) Coupled in vitro transcription-translation reaction. In vitro transcription/translation of the Strep-tagged GFP reporter protein was estimated by immunodetection. Reactions were supplemented with NAD + , Tre23 and Tri23, as indicated. In the last reaction, Tri23 was added 1 h after incubating the reaction with Tre23 (aft) and reaction proceeded for 2 h. ( D ) Tre23-Tri23 co-purification. Total cell lysates of E. coli BL21(DE3) cells producing FLAG-tagged Tri23 (Tri23 FL ) alone or co-producing Strep-tagged Tre23 (Tre23 ST ) (T) were subjected to purification on streptactin agarose beads followed by immunoprecipitation on anti-FLAG affinity gel (IP). Proteins were separated by SDS-PAGE and stained by Coomassie (upper panel) or immunodetected using anti-Strep and anti-FLAG antibodies (middle and bottom panels, respectively).

Article Snippet: C-terminally Strep-tagged Tre23 (Tre23ST ) was synthesized in a coupled in vitro transcription-translation reaction (PURExpress Kit, NEB).

Techniques: Fluorescence, Microscopy, Low Copy Number, Plasmid Preparation, In Vitro, Immunodetection, Copurification, Purification, Immunoprecipitation, SDS Page, Staining

Journal: Nucleic Acids Research

Article Title: Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA

doi: 10.1093/nar/gkab608

Figure Lengend Snippet: Tre23 ADP-ribosylates the 23S RNA of actively translating ribosomes. ( A and B ) Native PAGE (A) and SDS-PAGE (B) analyses of in vitro couple translation-transcription reaction supplemented with 6-biotin-17-NAD + ( biot NAD + ) and with Tre23, as indicated. Samples were stained with Coomassie blue (left panels) or detected using streptavidin-AP (right panels). Mass spectrometry analysis of the indicated band identified subunits of 30S and 50S ribosome (Ribos.). ( C ) RNA extracted from of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), Tre23 and Tri23, as indicated were stained with ethidium bromide (EtBr, left panel) or detected using streptavidin-AP conjugate (right panel). The positions of tRNAs, 16S and 23S ribosomal RNAs are indicated. ( D ) Western-blot analyses with anti-Strep antibodies of GFP-strepII in vitro transcription-translation in presence of ribosomes purified from wild-type E. coli cells carrying empty pNDM220 vector (Ø), or cells producing Tre23. Ribosomes (R) included in the coupled transcription-translation kit were used as control. Ribosome profile changes over time after toxin induction are shown in Supplementary Figure S2 . ( E ) RNA analyses of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), and Tre23, as indicated, using ribosomes purified from wild-type E. coli cells bearing empty vector (Ø), or cells producing Tre23. RNAs were stained with EtBr (left panel) or detected using streptavidin-AP (right panel). ( F ) EtBr staining (left panel) and Streptavidin-AP (right panel) RNA analyses of purified ribosomal RNAs (rRNA), or purified ribosomes alone or supplemented with translation factors (F) or translation reagents (tRNAs, amino acids, nucleotides; A), in presence of 6-biotin-17-NAD + ( biot NAD + ) and Tre23, as indicated.

Article Snippet: C-terminally Strep-tagged Tre23 (Tre23ST ) was synthesized in a coupled in vitro transcription-translation reaction (PURExpress Kit, NEB).

Techniques: Clear Native PAGE, SDS Page, In Vitro, Staining, Mass Spectrometry, Western Blot, Purification, Plasmid Preparation