The Expi293 mammalian cell expression system (Thermo Fisher Scientific, MA USA) with HE400 medium (GMEP, Fukuoka, Japan) was used to produce recombinant proteins. Expression vectors of VHH-CfaC fused heavy chain gene and light chain gene of M291 antibody were simultaneously transfected to the cell using PEI MAX reagent (Polyscience Inc., PA USA) for CfaC-IgG expression. Expression vectors of CfaN fused VH-CH1 gene and the light chain gene of trastuzumab for Fab-CfaN were transfected similarly for CfaC-IgG expression. The supernatants were collected and applied to the purification columns 7 days after culture. Protein A column (UNOsphere SUPrA, Biorad, CA USA) was used for the CfaC-IgG purification and Ni–NTA column (FUJIFILM Wako chemicals, Osaka, Japan) for the Fab-CfaN purification.
Ni nta column
Manufactured by Fujifilm
Sourced in Japan
The Ni-NTA column is a laboratory equipment used for protein purification. It consists of a matrix of nickel-nitrilotriacetic acid (Ni-NTA) that is used to selectively bind and purify recombinant proteins containing a histidine-tag. The column facilitates the separation and isolation of the target protein from the rest of the sample components.
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Lab products found in correlation
3 protocols using ni nta column
1
Recombinant Cfa-Fusion Protein Expression
2
Recombinant Indicator Protein Purification
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Recombinant indicators, N-terminally tagged with polyhistidine, were expressed in E. coli (JM109(DE3)) for 4-5 days at 22 °C with gentle shaking at 130 rpm. The E. coli cells were lysed using B-PER. Subsequently, the recombinant indicator was purified using a Ni-NTA column (Wako) and underwent buffer exchange employing a Microsep Advance Centrifugal Device with a 30 K MWCO (PALL). The buffers used for the purified indicators were exchanged with the appropriate ones for GECIs 6 , iGluSnFR 31 , and GINKO 32 . Protein concentrations were determined as previously reported 13 . Fluorescence spectra were recorded using a fluorescence spectrophotometer (F-7000, Hitachi), and absorption spectra were recorded using a microplate reader (SpectraMax iD3, Molecular Devices). For kinetic analysis, the dissociation dynamics were measured using a stopped-flow device (SFS-853 and FP-8000, JASCO), as previously reported 9 . Briefly, fluorescence signal changes by depletion of Ca 2+ from the indicators through rapid mixing with 10 mM EGTA were recorded every 5 ms up to 30 s, then were fitted to a single exponential curve to determine the k off value using OriginPro 9 software (LightStone). The k on value was calculated using the independently determined K d through the following equation:
3
Purification and Characterization of R-FlincA
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As the purification of R-FlincA from E. coli and mammalian cells was not successful, we expressed N-terminal polyhistidine-tagged R-FlincA in D. discoideum cells cultured in HL5 medium at 22 °C. Cells (8 × 108) were starved in the development buffer (5 mM Na2HPO4, 5 mM KH2PO4, 1 mM CaCl2, and 2 mM MgCl2 at pH 6.4) for 12 hours, and then lysed using n-octyl-1-thio-β-D-glucopyranoside. The recombinant indicator was purified using a Ni-NTA column (Wako), followed by buffer exchange to Hepes buffer (pH 7.4) using a MicrosepTM advance centrifugal device 30 K MWCO (PALL). For unknown reasons, the amount of purified R-FlincA was not enough for the absorbance measurements, but it was sufficient for the measurement of excitation and emission spectra. Emission spectra (550–700 nm with a 5-nm bandwidth) upon 530 nm excitation (Xenon, 150 W) and the excitation spectra (450–590 nm) with 635 nm emission were measured using a fluorescence spectrophotometer (F-4500, Hitachi). pH titrations were performed by using a series of buffers prepared with pHs ranging from 4 to 11 as described8 (link).
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