Recombinant Protein Expression and Purification

Bacterial cells were cultured in Luria Bertani (LB) broth at 37°C to an optical density of OD₆₀₀ = 0.8. Protein production was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (AppliChem GmbH, Germany). Bacteria were further incubated at 16°C for 18 h. Protein extraction and purification were conducted as previously described (Schubert et al., 2012 (link)). HIS-tagged proteins were purified by metal-affinity chromatography using Ni-NTA resins (Qiagen). Protein concentration was estimated by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and the purity of CCL2 and CCL2-Y92A was examined by SDS-PAGE.

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Moradi A., El-Shetehy M., Gamir J., Austerlitz T., Dahlin P., Wieczorek K., Künzler M, & Mauch F. (2021). Expression of a Fungal Lectin in Arabidopsis Enhances Plant Growth and Resistance Toward Microbial Pathogens and a Plant-Parasitic Nematode. Frontiers in Plant Science, 12, 657451.

Publication 2021

isopropyl β-D-1-thiogalactopyranoside (IPTG) Ni-NTA resins Pierce BCA Protein Assay Kit

Affinity chromatography Assay Bacteria Ccl2 Cells Metal Protein Resins Sds page

Corresponding Organization :

Other organizations : University of Fribourg, Tanta University, Universitat Jaume I, BOKU University, Agroscope

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Variable analysis

independent variables

Addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for protein production induction

dependent variables

Optical density (OD) at 600 nm
Protein concentration
Purity of CCL2 and CCL2-Y92A as examined by SDS-PAGE

control variables

Culturing bacterial cells in Luria Bertani (LB) broth
Incubation temperature of 37°C to reach OD600 = 0.8
Subsequent incubation at 16°C for 18 h after IPTG induction
Protein extraction and purification methods as previously described (Schubert et al., 2012)

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