Synthesis data by WEPRO7240G and WEPRO7240H
Each proteins were synthesized by Protemist DTII with 6ml Bi-layer reaction of WEPRO7240G or WEPRO7240H
lane C:crude protein, lane E : purified protein fraction
GST-Tag purification：Resin, Glutathione Sepharose 4B (Cytiva) Equilibration buffer & Wash buffer: PBS
Elution buffer : PBS
His-Tag：Resin, Ni Sepharose High Performance (Cytiva)
Equilibration buffer & Wash buffer: 20mM Phosphate buffer, 0.3 M NaCl, 50mM Imiadzole, pH7.5
Elution buffer : 20mM Phosphate buffer, 0.3 M NaCl, 500mM Imiadzole, pH7.5
Synthesis comparison data by WEPRO®7240, WEPRO®7240G and 7240H
Synthesis comparison data of WEPRO7240 with WEPRO7240G and 7240H
Synthesis and purification using WEPRO7240G and 7240H excludes wheat germ-derived proteins other than the target protein (*) found when using WEPRO7240.
A: Human-derived proteins with GSTs added (◀) are WEPRO7240(1) and WEPRO7240G(2, respectively, followed by affinity purification.
B: Virus-derived proteins (◀) to which His was added were synthesized using WEPRO7240(1) and WEPRO7240H(2), respectively, followed by affinity purification.
Membrane Protein synthesis:
Proteomicelle : Effect of detergent for membrane protein expression
Digitonin (non-ionic steroid derivative) increased both the yield and the solubility of the 7 solubilized proteins.
Brij-35 and Brij-58 (polyoxyethylene alkyl-ethers) increased the solubility of the 7 solubilized proteins without decreasing their protein yields at tested concentrations. Tween-20, another polyoxyethylene alkyl-ether, had positive effect on the solubility of 5 proteins.
Triton X-100 and Nonidet P-40 (polyethylene glycol derivatives) showed either positive or negative effects on yield and solubility depending on proteins.
DDM and ß-OG (alkyl glucosides), CHAPS (zwittergenic steroid derivative), and DPC (mono-chain phosphocoline) little improved the solubility of the proteins tested.
Solubility testing : the effect of detergents for solubility of GPR84
3 μl of total and soluble fractions were analyzed by SDS-PAGE.
T: total fraction, S: soluble fraction
◀: synthesized protein
ProteoLiposome :
Proteoliposome kit dedicated for membrane proteins like GPCRs, ion channels and transporters
Various GPCRs including Class A, B and C could be synthesized as proteoliposome with 2.5 mL reaction volume of BD (Bi-Layer Dialysis) reaction format.
H. Takeda et al. (2015) Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay Scientific Reports Rep.5, 11333
Effect of synthesis reaction temperature on the amount of synthesis by WEPRO7240
The mRNA obtained from the cloned template DNA was transcribed according to the usual protocol, and then translated at 4°C, 10°C, 15°C, and 25°C. After the reaction, the GFP fluorescence was measured. After the reaction, GFP fluorescence was measured.The results showed that the best yield was obtained at 15°C for 20 hr. The synthesis reaction was also observed at 4°C, and the yield increased by extending the reaction time, indicating the possibility of improving the properties of the synthesized protein by lowering the reaction temperature for thermosensitive targets.
