Pd minitrap g 25 column

Benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide (Z-Phe-Arg-MCA) and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-Agmatine (E-64) were purchased from Peptide Institute Inc., Osaka, Japan, DMEM medium, GSSG, DTT (Wako, Japan), Lipofectamine LTX and PLUS (Invitrogen, Oslo, Norway), Quickchange II site-directed mutagenesis kit (Agilent Technologies, CA, USA), Primestar mutagenesis kit (Takara, Japan), plasmid pFUSE-hlgG2-Fc1 (InvivoGen, CA), Toyopearl HW-50 (Tosoh Co., Tokyo, Japan), PD MiniTrap G-25 column (GE Healthcare, UK), and Dynabeads TALON (Invitrogen, Oslo, Norway).

TLR7_lig- and TLR4_lig-Pp3 were labeled with Alexa Fluor
647 NHS Ester (succinimidyl ester) (TLR7_lig- and TLR4_lig-Pp3-Alexa Fluor 647) according to the manufacturer’s
instructions (Thermo Fisher Scientific). After 1 h of incubation with
constant stirring in the dark, the uncoupled free Alexa Fluor 647
was removed by gel filtration column (PD MiniTRap G-25 column, GE
Healthcare, Chicago, IL) with PBS. The TLR7_lig- and TLR4_lig-Pp3-Alexa Fluor 647 were concentrated with a Nanodrop 2000
spectrophotometer (Thermo Fisher Scientific).

For purification of StrepII-tagged catalase cells of strain ELF7/pLUMB38 were grown and disrupted as described for ELF7/pLUF15 (His-tagged catalase). For preparation of cytoplasmic fraction 100 mm Tris·HCl, pH 8.0, 150 mm NaCl was used instead of phosphate buffer. Gravity flow affinity chromatography was carried out on a 0.5-ml Strep-Tactin column (IBA) that was prepared according to the manufacturer's instructions. After loading the sample onto the column it was washed thrice with 0.5 ml of buffer W (100 mm Tris·HCl, pH 8.0, 150 mm NaCl) followed by elution of the KatA-Strep fusion protein with 6 × 0.25 ml of buffer E (100 mm Tris·HCl, pH 8.0, 150 mm NaCl, 2.5 mm desthiobiotin). The presence and purity of catalase protein in fractions collected during washing and elution was determined by SDS-PAGE and immuno-blot. For further analysis of the purified protein the buffer was changed to 50 mm KPO₄, pH 8.0 using a PD MiniTrap G-25 column (GE Healthcare).

The concentrated apo-DddW was reconstituted as follows. 500 μL of 199 μM apo-protein was incubated with 500 μM Fe(NH₄)₂(SO₄)₂ for 1 h followed by desalting in a GE PD MiniTrap G-25 column. The column was washed in a buffer containing 50 mM HEPES pH 7.5, 10 mM NaCl. The sample was loaded and allowed to enter the resin before more solution of buffer was added. The reconstituted protein was eluted from the column and 0.5 mL aliquots were collected during the gravity elution. The eluted protein was analyzed for metal content consistently yielding a holo-enzyme with 85–90 mol percent iron.

HEK293T cells transfected with an expression vector for C-terminally histidine (His)-tagged Got1l1 (pEB-Got1l1-His) and an empty vector (pEB6) were collected from two Petri dishes (10 cm diameter) each for the preparation of recombinant Got1l1 and control cell extract, respectively. The cells were sonicated in 400 μl of lysis buffer consisting of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 % Triton X-100, and protease inhibitors cocktail lacking EDTA (nacalai tesque, Kyoto, Japan), and centrifuged at 20,000×g for 30 min. The supernatant was incubated with Ni-Sepharose 6 fast flow resin (GE Healthcare) for 4 h at 4 °C. After the resin was washed five times with wash buffer (PBS containing 10 mM imidazole and 0.5 % Triton X-100), proteins were eluted with 500 μl of elution buffer (PBS containing 1 M imidazole). The eluates were immediately passed through a PD MiniTrap G-25 column (GE Healthcare) equilibrated with PBS. Then, a 100-μl aliquot of the protein solution was mixed with the same volume of the reaction mixture consisting of 50 mM Tris–HCl (pH 7.5), 40 μM PLP, 10 mM l-aspartate, and 10 mM α-ketoglutarate. After 4 h incubation at 37 °C, the reaction was terminated by adding 200 μl of 0.2 M TCA. The formation of d-aspartate, d-glutamate, and l-glutamate was examined by HPLC as described above.