Recombinant Protein Expression and Purification

Recombinant proteins were expressed using the E. coli (BL21, Arctic strain) system after induction by 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 13°C overnight as described elsewhere.34 (link),37 (link),38 (link) Soluble Hisx6-tagged proteins were purified using the Cobalt resins (Thermo Fisher Scientific), while GST and GST-tagged protein were isolated using the Glutathione Sepharose 4 Fast Flow medium (GE Healthcare Life Sciences) according to the instructions of the manufacturers. The tag-free S-αTSR protein was purified by a two-step procedure, including a precipitation of the target protein from the bacteria lysate using ammonium sulfate [(NH₄)₂SO₄], and an ion exchange chromatography (see below).35 (NH₄)₂SO₄ at 1.6 M end concentration was used to effectively precipitate the tag-free S-αTSR protein at room temperature (20–22°C).

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Xia M., Vago F., Han L., Huang P., Nguyen L., Boons G.J., Klassen J.S., Jiang W, & Tan M. (2023). The αTSR Domain of Plasmodium Circumsporozoite Protein Bound Heparan Sulfates and Elicited High Titers of Sporozoite Binding Antibody After Displayed by Nanoparticles. International Journal of Nanomedicine, 18, 3087-3107.

Publication 2023

Cobalt resins Glutathione Sepharose 4 Fast Flow medium

Ammonium sulfate Bacteria lysate Cobalt E coli Glutathione Ion exchange chromatography Protein Protein target Recombinant proteins Resins Sepharose Strain

Corresponding Organization :

Other organizations : Cincinnati Children's Hospital Medical Center, Purdue University West Lafayette, University of Alberta, University of Cincinnati Medical Center

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Variable analysis

independent variables

Induction by 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG)
Temperature of 13°C overnight

dependent variables

Expression of recombinant proteins
Purification of soluble His-tagged and GST-tagged proteins
Purification of tag-free S-αTSR protein

control variables

E. coli (BL21, Arctic strain) expression system

controls

Positive control: Not specified
Negative control: Not specified

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