Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli
Yoshinori Hirano,
Yong Gao,
Dhirendra Simanshu,
Daniel Stephenson,
Ngoc Vu,
Lucy Malinina,
Charles Chalfant,
Dinshaw Pate,
Rhoderick Brown
Affiliations
Yoshinori Hirano
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, U.S.AGraduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama, Japan
Yong Gao
Hormel Institute, University of Minnesota, Austin, MN, U.S.A.
Dhirendra Simanshu
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, U.S.A
Daniel Stephenson
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University Medical Center, Richmond, VA, U.S.A.Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, U.S.A.
Ngoc Vu
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University Medical Center, Richmond, VA, U.S.A.
Lucy Malinina
Hormel Institute, University of Minnesota, Austin, MN, U.S.A.
Charles Chalfant
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University Medical Center, Richmond, VA, U.S.A.Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, U.S.A., Research Service, James A. Haley Veterans Hospital, Tampa, FL, U.S.A., The Moffitt Cancer Center, Tampa, FL, U.S.A.
Dinshaw Pate
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, U.S.A
Rhoderick Brown
Hormel Institute, University of Minnesota, Austin, MN, U.S.A.
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)
