TNF-Fc, HF-TNFSF10/TRAIL and FASLG-Fc were expressed and purified as described before [15 (link),57 (link),58 (link)]. In short, the cDNAs encoding the extracellular portion of human TNF (AA78–233) or human FASLG/CD95L (AA117–281) were fused at the N terminus to the constant region (Fc) of human IgG1 (CH2-CH3, AA102–329), preceded by the TNFRSF10B signal peptide (AA1–55). The expression vector pcDNA3.1 (Invitrogen, V79020) was transfected in HEK293T cells and the supernatants containing the Fc-tagged proteins were used for the experiments shown. HF-TNFSF10/TRAIL was produced by cloning the extracellular portion of human TNFSF10/TRAIL (AA95–281) in the pQE32 vector (Qiagen, N32915) preceded by a FLAG and 6×His tag. The protein was expressed in M15 bacteria (XL Biotech, CHC00032) and purified using Ni-NTA (Cytiva, 29048631).
Ni nta
Manufactured by Cytiva
Ni-NTA is a nickel-nitrilotriacetic acid resin that is commonly used in affinity chromatography for the purification of recombinant proteins containing a histidine (His) tag. The Ni-NTA resin binds to the His-tagged proteins, allowing them to be separated from other components in the sample.
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3 protocols using ni nta
1
Purification of Fc-tagged TNF and FASLG
2
Purification and Characterization of Acidaminococcus sp. dCas12a
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The Acidaminococcus sp dCas12a protein was expressed in Escherichia coli BL21 and purified by chromatography on Ni-NTA (Cytiva), HiTrap SP HP (Cytiva), and HiLoad Superdex 200 16/60 (Cytiva) columns and determined purity through polyacrylamide gel electrophoresis. The 55 nt guide RNA (gRNA) was transcribed in vitro and then purified by TRIzol (Invitrogen) and verified integrity through denaturing urea polyacrylamide gel electrophoresis.
3
Production of Recombinant CD30 and CD5 Proteins
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The CD30 and CD5 antigen extracellular domains were amplified from the Karpas cell cDNA by PCR and cloned into the pcDNA3.1 expression vector ligated with the His tag. The CD30 and CD5 expression plasmid was transfected into HEK293F cells with PEI reagent. Cells were then given the supplement reagent (Chinese hamster ovary [CHO] PFF05, Shanghai OPM Biosciences Co., Ltd) twice after 24 h and after 96 h. About 7 days after transfection, the supernatant of the cell culture was collected. The supernatants were incubated with Ni-NTA (Nitrilotriacetic acid) agarose (Cytiva) to enrich His-tagged CD30 and CD5 antigen extracellular domain proteins, followed by protein elution with Tris-NaCl buffer containing imidazole. The concentration of protein was determined by BCA assay. The scFv -Fc or Nb-Fc fragments were cloned into the pcDNA3.1 expression vector. After transfection into HEK293F cells, the culture supernatant was purified and concentrated by protein A, and the scFv/Nb-Fc protein was used for subsequent ELISA experiments.
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