Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli

Yoshinori Hirano; Yong-Guang Gao; Dhirendra K Simanshu; Daniel J Stephenson; Ngoc T Vu; Lucy Malinina; Charles E Chalfant; Dinshaw J Patel; Rhoderick E Brown

doi:10.21769/BioProtoc.3906

Purification of Cytosolic Phospholipase A₂α C2-domain after Expression in Soluble Form in Escherichia coli

Bio Protoc. 2021 Feb 5;11(3):e3906. doi: 10.21769/BioProtoc.3906.

Authors

Yoshinori Hirano^{1

2}, Yong-Guang Gao³, Dhirendra K Simanshu¹, Daniel J Stephenson^{4

5}, Ngoc T Vu⁴, Lucy Malinina³, Charles E Chalfant^{4

5

6

7}, Dinshaw J Patel¹, Rhoderick E Brown³

Affiliations

¹ Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, U.S.A.
² Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama, Japan.
³ Hormel Institute, University of Minnesota, Austin, MN, U.S.A.
⁴ Department of Biochemistry and Molecular Biology, Virginia Commonwealth University Medical Center, Richmond, VA, U.S.A.
⁵ Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, U.S.A.
⁶ Research Service, James A. Haley Veterans Hospital, Tampa, FL, U.S.A.
⁷ The Moffitt Cancer Center, Tampa, FL, U.S.A.

Abstract

Previous expression/purification strategies for cytosolic phospholipase A₂α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented herein describes an effective method for the expression of cytosolic phospholipase A₂α C2-domain in soluble form in E. coli and subsequent purification to homogeneity. This protocol, which utilizes a cleavable 6xHis-SUMO tag, has recently been used to gain insights into the structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A₂α ( Hirano et al., 2019 ).

Keywords: Cytosolic phospholipase A2α; Soluble expression in E. coli; 6xHis-SUMO tag; C2-domain purification.

Grants and funding

P30 CA008748/CA/NCI NIH HHS/United States