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Article

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

by
Thi Kieu Oanh Nguyen
1,
Thi Luong Vu
1,
Minh Quan Nguyen
1,
Huynh Kim Khanh Ta
1,
Kyoung Sun Park
2,
Soo Hyeon Kim
2,
Chong Jai Kim
3,
Yeon Jin Jang
1 and
Han Choe
1,*
1
Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea
2
Wide River Institute of Immunology, Seoul National University College of Medicine, Seoul 25159, Korea
3
Department of Pathology, Asan-Minnesota Institute for Innovating Transplantation, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(10), 5267; https://doi.org/10.3390/ijms22105267
Submission received: 14 April 2021 / Revised: 6 May 2021 / Accepted: 15 May 2021 / Published: 17 May 2021
(This article belongs to the Section Biochemistry)
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Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.
Keywords: hGM-CSF; MBP; PDI; ClearColi hGM-CSF; MBP; PDI; ClearColi

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MDPI and ACS Style

Nguyen, T.K.O.; Vu, T.L.; Nguyen, M.Q.; Ta, H.K.K.; Park, K.S.; Kim, S.H.; Kim, C.J.; Jang, Y.J.; Choe, H. Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain. Int. J. Mol. Sci. 2021, 22, 5267. https://doi.org/10.3390/ijms22105267

AMA Style

Nguyen TKO, Vu TL, Nguyen MQ, Ta HKK, Park KS, Kim SH, Kim CJ, Jang YJ, Choe H. Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain. International Journal of Molecular Sciences. 2021; 22(10):5267. https://doi.org/10.3390/ijms22105267

Chicago/Turabian Style

Nguyen, Thi Kieu Oanh, Thi Luong Vu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Kyoung Sun Park, Soo Hyeon Kim, Chong Jai Kim, Yeon Jin Jang, and Han Choe. 2021. "Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain" International Journal of Molecular Sciences 22, no. 10: 5267. https://doi.org/10.3390/ijms22105267

APA Style

Nguyen, T. K. O., Vu, T. L., Nguyen, M. Q., Ta, H. K. K., Park, K. S., Kim, S. H., Kim, C. J., Jang, Y. J., & Choe, H. (2021). Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain. International Journal of Molecular Sciences, 22(10), 5267. https://doi.org/10.3390/ijms22105267

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