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Expression and Purification of LpPLA2 for Crystallography
Author(s) -
Matico Rosalie Elizabeth,
Myers Linda,
Taylor David,
Sweitzer Sharon,
Elkins Patricia,
Smallwood Angela,
Pietrak Beth,
Schwartz Benjamin,
McCormick Patricia,
Johanson Kyung
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb236
Subject(s) - chemistry , cleavage (geology) , sepharose , biochemistry , enzyme , biology , paleontology , fracture (geology)
Lipoprotein‐associated lipase (LpPLA2) is a 45kD calcium independent lipase secreted by macrophages, T cells, and mast cells in atherosclerotic plaques. LpPLA2 is mainly associated with ApoB on LDL particles and is responsible for hydrolyzing the sn‐2 chain of oxidatively modified phospholipids resulting in lysophatidylcholine and non‐esterified fatty acids ‐ both inflammatory mediators. Crystallography of LpPLA2 would be beneficial for inhibitor optimization. LpPLA2 was expressed by baculovirus with a dual N‐terminal his/Streptavidin binding protein (SBP) tag and a TEV cleavage site engineered prior to residues 47‐429. No signal sequence was engineered into the construct; however, active LpPLA2 was detected at high levels in both the medium (~35mg/L) and cells (~15mg/L). His/SBP/TEV(GS)LpPLA2 (47‐429) was captured from the medium by Ni Sepharose, the his/SBP/TEV(GS)LpPLA2(47‐429) in the imidazole eluate was captured onto Streptavidin resin and (GS)LpPLA2 (47‐429) was released by on column TEV cleavage. (GS)LpPLA2(47‐429) was further purified by Blue‐Sepharose. Mono Q was used for high resolution chromatography and detergent exchange. Final product demonstrated full active site accessibility by titration with GSK184919A which acylates the active site serine with a dimethylamido group resulting in a +71Da mass shift. A surface cysteine was susceptible to cysteinylation and this appeared to have no impact on activity. Crystals of the final product formed within a day and the apo‐structure was solved to ~2.0? resolution.