TY - JOUR
T1 - Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma
AU - Olson, Tien L.
AU - Zhang, Shangji
AU - Labban, Dillon
AU - Kaschner, Emily
AU - Aceves, Manuel
AU - Iyer, Srivatsan
AU - Meza-Aguilar, Jose Domingo
AU - Zook, James D.
AU - Chun, Eugene
AU - Craciunescu, Felicia M.
AU - Liu, Wei
AU - Shi, Chang Xin
AU - Stewart, A. Keith
AU - Hansen, Debra T.
AU - Meurice, Nathalie
AU - Fromme, Petra
N1 - Publisher Copyright:
© 2021
PY - 2021/9
Y1 - 2021/9
N2 - Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and β-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.
AB - Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and β-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.
KW - Crystallography
KW - G-protein coupled receptor kinase 6
KW - Multiple myeloma
KW - Palmitoylation
KW - Structure-guided drug design
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U2 - 10.1016/j.pep.2021.105890
DO - 10.1016/j.pep.2021.105890
M3 - Article
C2 - 33971243
AN - SCOPUS:85106932337
SN - 1046-5928
VL - 185
JO - Protein Expression and Purification
JF - Protein Expression and Purification
M1 - 105890
ER -