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Expression and Purification of Rickettsial Outer Membrane Protein B
Author(s) -
Choi BokEum,
Abeykoon A,
Chao C C,
Wang G.,
Gucek M.,
Ching W M.,
Chock P B.,
Buchanan S.,
Yang D C H.
Publication year - 2015
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.29.1_supplement.714.2
Subject(s) - chemistry , bacterial outer membrane , circular dichroism , methylation , guanidinium chloride , methyltransferase , biochemistry , denaturation (fissile materials) , urea , escherichia coli , chromatography , biophysics , biology , enzyme , nuclear chemistry , gene
Outer membrane protein B is an autotransporter protein present in all rickettsial species. Methylation of OmpB has been implicated in bacterial virulence. Two distinct types of lysine methyltransferases (PKMT1 and PKMT2) have been characterized using rOmpB as substrates. Structural studies of OmpB‐MTase complexes are necessary to better understand the mechanistic action of these unique MTases, but have been hampered by poor expression, purification and solubility of OmpB. Using codon‐optimized rOmpB fragments remarkably improved expression in E. coli . Denaturation with 6M guanidinium chloride appreciably elevated the yield of purified 6xHis‐tagged fragments using Ni‐NTA. Renaturation with decreasing concentrations of urea invariably produced insoluble aggregates. Partially refolded rOmpB fragments were then subjected to refolding in the second Ni‐NTA column. Our results indicate that the two‐step Ni‐NTA process produced highly pure and soluble rOmpB. The presence of multiple methylation sites and the circular dichroism spectra of the purified rOmpB fragments indicate that the Ni‐NTA resin facilitates surface‐mediated refolding of rOmpB. These expression, purification and refolding procedures allow us to overcome the obstacles to obtain a large quantity of rOmpB for crystallographic studies. The results also implicate a potential role of in vivo surface‐mediated OMP protein folding.