Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study.
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH2 to produce PGD2. Three cysteine residues, Cys65, Cys89, and Cys186 in L-PGDS, are conserved among all species, of which Cys89 and Cys186 residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.
Liu
Jiafu
J
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
Lin
Kejiang
K
Guo
Chenyun
C
Gao
Hongchang
H
Yao
Yihe
Y
Lin
Donghai
D
D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000502:physiology*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054834:Lipocalins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D054834:Lipocalins; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D003545:Cysteine