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

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

Yoshinori Hirano  1   2 , Yong-Guang Gao  3 , Dhirendra K Simanshu  1 , Daniel J Stephenson  4   5 , Ngoc T Vu  4 , Lucy Malinina  3 , Charles E Chalfant  4   5   6   7 , Dinshaw J Patel  1 , Rhoderick E Brown  3
Affiliations

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

Yoshinori Hirano et al. Bio Protoc. .

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.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Chromatogram of gel filtration purification (Step 15 in the Procedure).
Y-axis represents absorbance at 280 nm whereas the X-axis represents elution time. The arrow corresponds to the elution peak for monomeric cPLA2α C2-domain (16-140; 14.3 kDa).
Figure 2.
Figure 2.. SDS-PAGE analysis of purified chicken cPLA2α C2-domain (16-140).
The protein load for purified C2-domain equals 1-2 μg.
See this image and copyright information in PMC

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