Human Cell-Free Protein Expression System from Takara (Cat. #3281) was used to determine the effect of SidH on host protein synthesis. 0.5 µM of purified proteins (SidH FL/SidI/HM/SidHParis) were mixed with different components of the kit including a plasmid which encodes for β-galactosidase in a 20 µl reaction. The reaction mix was incubated at 30 °C for different time points. 2 µl of the reaction mix was incubated with 0.7 M of o-nitrophenyl- β-D-galactopyranoside (ONPG) (MERCK; Cat. #73660) 37 °C for 30 min in cleavage buffer (60 mM Na2HPO4.7H2O, 40 mM NaH2PO4.H2O, 10 mM KCl, 1 mM MgSo4.7H2O, pH 7.0, 0.4 M β-Mercaptoethanol). The reaction was stopped by adding 0.5 M of Sodium Carbonate. Absorbance was measured at 420 nm using the plate reader and graphs were plotted. This experiment was done in triplicates.
Human cell free protein expression system
Manufactured by Takara Bio
Sourced in Japan
The Human Cell-Free Protein Expression System is a laboratory tool that allows for the synthesis of proteins from human cell extracts in a controlled in vitro environment. It provides a platform for the production and study of human proteins without the need for living cells.
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8 protocols using human cell free protein expression system
1
Investigating SidH's Impact on Protein Synthesis
2
In Vitro Transcription and Translation Assay
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Human Cell-Free Protein Expression System (TaKaRa Bio Inc., Shiga, Japan) was used for in vitro transcription and translation. The procedure for the preparation of the mixture and the translation and transcription were conducted according to the instructions with several modifications as shown below. Briefly, 9 µL of cell lysate, 6 µL of Mixture-1, and 1 µL of Mixture-2 in the system were mixed with 0.5 µL of a solution containing a trichothecene at various concentrations or solvent. After incubation for 10 min at room temperature, 2 µL of Mixture-3, 1 µL of T7 RNA polymerase solution (200 U/µL), and 0.5 µL of a plasmid (0.3 μg/mL) containing β-galactosidase gene in the system were added, followed by incubation at 32 °C for 1, 2, or 3 h.
3
PDPK1 Variants Binding Assay
4
Protein Expression Assay for Hint1 and Caren
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An in vitro translation assay was performed using a Human Cell-Free Protein Expression System (Takara Bio Inc), according to the manufacturer’s instructions. In brief, cDNA encoding C-terminally HA-tagged murine Hint1 was cloned into the pUC-T7-IRES vector (Takara Bio Inc), and Caren, antisense Caren, and Caren fragment A–E cDNAs were cloned into pSP72 (Promega, Madison, WI, USA). The plasmid encoding Hint1 and either the pSP72 vector or plasmids encoding Caren, antisense Caren or one of Caren fragments A–E were incubated with T7 RNA polymerase in human cell lysates at 37 °C for 2 h. Samples were then subjected to western blotting analysis. In some experiments, in vitro-transcribed Caren was incubated at 94 °C for 2 min and snap-cooled on ice for denaturation66 (link). Plasmids encoding Hint1 plus either native or denatured Caren were incubated with T7 RNA polymerase in human cell lysates at 37 °C for 2 h and then subjected to western blotting analysis.
5
Analyzing mRNA Nano-Lantern Translation
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Human Cell-Free Protein Expression System (Takara, Dalian, China) was used to analyze the translation ability of mRNA nano-lantern according to its protocol. Briefly, naked Smad4 mRNA, mRNA nano-lantern (3 bs, 5 bs, 7 bs) with the same concentration of 25 ng/μL was incubated in the solution containing HeLa Lysate, Accessory Proteins, Reaction Mix for 6 h at 30 °C, respectively. Western blot analysis was adopted to detect the expression of the targeted protein.
6
Protein Interaction Assay with Nurr1, PARP1, and MLYCD
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For pull-down assays, FLAG-Nurr1, HA-PARP1 and FLAG-MLYCD were translated in vitro using Human Cell-Free Protein Expression System (Takara Bio, Tokyo, Japan). FLAG-tagged proteins were mixed with HA-PARP1 for 1h at room temperature and then immunoprecipitated with Anti-FLAG M2 affinity agarose gel (Sigma-Aldrich, A2220). Purified proteins were subjected to western blotting.
7
LRRK2 Kinase Inhibition Assay
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A human, cell-free protein expression system from Takara Bio Inc (Japan) was used according to the manufacturer's instructions. Briefly, a β-galactosidase reporter (150 ng) was incubated with 60 nM GST-G2019S-LRRK2 or GST alone for 1.5 hours at 32°C. β-galactosidase expression was measured using colorimetric detection (Abs 420 nm , using a Synergy H1 plate reader (Biotek) from samples in a 96-well plate. Absorbance at 420 nm was measured using the Synergy H1 Hybrid Reader (Biotek).
8
PDPK1 Variants Binding Assay
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In vitro transcription and translation of PDPK1 variants was performed using the Takara Human Cell-Free Protein Expression System (Takara #3281) following the manufacturer’s protocol. Yield of the in vitro reaction was quantified by western blotting alongside a FLAG-tagged standard. For binding reactions each in vitro reaction was incubated with 20 mg of 6xHis-SUMO-WDR5 (22–334) bound to NTA-Ni beads. After combining PDPK1 and WDR5, inputs were removed and binding reactions were performed in a 500μl volume in Kischkel buffer for 2 hours at 4°C. Beads were washed with 1 mL cold Kischkel buffer four times for two minutes and transferred to new tubes before the last wash. Samples were eluted by boiling in SDS sample buffer supplemented with β-mercaptoethanol and analyzed by western blotting.
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