Secretagogin Purification and Quality Control Strategies for Biophysical and Cell Biological Studies
Secretagogin (SCGN) has recently gained attention due to its modulatory effect on insulin/CRH secretion and function. However, a large pool of speculated SCGN functions remains unexplored. A major deficiency is the lack of knowledge about the biological functions of extracellular SCGN. We here describe convenient methods for the scalable production of His-tagged and untagged mouse SCGN. The protocol is optimized to remove endotoxins, and thus the protein is suited for biological applications such as cell culture treatment or animal injections. We also outline expedient methods to check the purity of SCGN preparation for biological applications.
Key wordsSecretagogin Protein purification Calcium sensor protein His-tag purification Chromatography Circular dichroism
This work was supported partially by CSIR and DBT to YS. AKS was supported by the Senior Research Fellowship, University Grant Commission, and RK is the recipient of CSIR-GATE fellowship. Authors acknowledge occasional help from HC Amrutha during the preparation of this manuscript. Anand Kumar Sharma and Radhika Khandelwal contributed equally to this work.
- 4.Romanov RA, Alpár A, Zhang MD, Zeisel A, Calas A, Landry M, Fuszard M, Shirran SL, Schnell R, Dobolyi A, Oláh M, Spence L, Mulder J, Martens H, Palkovits M, Uhlen M, Sitte HH, Botting CH, Wagner L, Linnarsson S, Hökfelt T, Harkany A (2014) A secretagogin locus of the mammalian hypothalamus controls stress hormone release. EMBO J 34:36–54CrossRefGoogle Scholar
- 6.Malenczyk K, Girach F, Szodorai E, Storm P, Segerstolpe Å, Tortoriello G, Schnell R, Mulder J, Romanov RA, Borók E, Piscitelli F, Di Marzo V, Szabó G, Sandberg R, Kubicek S, Lubec G, Hökfelt T, Wagner L, Groop L, Harkany T (2017) A TRPV1-to secretagogin regulatory axis controls pancreatic β-cell survival by modulating protein turnover. EMBO J 36(14):2107–2125CrossRefGoogle Scholar
- 7.Sharma AK, Khandelwal R, Chadalawada S, Ram NS, Raj A, Jerald MK, Sharma Y. SCGN administration prevents insulin resistance and diabetic complications in high-fat diet-fed animals. bioRxiv. 2017. https://doi.org/10.1101/189324
- 8.Hansson SF, Zhou AX, Vachet P, Eriksson JW, Pereira MJ, Skrtic S, Jongsma Wallin H, Ericsson-Dahlstrand A, Karlsson D, Ahnmark A, Sörhede Winzell M, Magnone MC, Davidsson P (2018) Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction. PLoS One 13(4):e0196601CrossRefGoogle Scholar