title: Expression, purification, and characterization of formaldehyde dehydrogenase from Pseudomonas aeruginosa.
abstract: As a member of zinc-containing medium-chain alcohol dehydrogenase family, formaldehyde dehydrogenase (FDH) can oxidize toxic formaldehyde to less active formate with NAD(+) as a cofactor and exists in both prokaryotes and eukaryotes. Most FDHs are well known to be glutathione-dependent in the catalysis of formaldehyde oxidation, but the enzyme from Pseudomonas putida is an exception, which is independent of glutathione. To identify novel glutathione-independent FDHs from other bacterial strains and facilitate the corresponding structural and enzymatic studies, high-level soluble expression and efficient purification of these enzymes need to be achieved. Here, we present molecular cloning, expression, and purification of the FDH from Pseudomonas aeruginosa, which is a Gram-negative pathogenic bacterium causing opportunistic human infection. The FDH of P. aeruginosa shows high sequence identity (87.97%) with that of P. putida. Our results indicated that coexpression with molecular chaperones GroES, GroEL, and Tig has significantly attenuated inclusion body formation and improved the solubility of the recombinant FDH in Escherichiacoli cells. A purification protocol including three chromatographic steps was also established to isolate the recombinant FDH to homogeneity with a yield of ∼3.2 mg from 1L of cell culture. The recombinant P. aeruginosa FDH was properly folded and biologically functional, as demonstrated by the mass spectrometric, crystallographic, and enzymatic characterizations of the purified proteins.
authors.affiliation: R&D Department, Novoprotein Scientific Inc (Shanghai), Room 202, Building 2, 720 Cailun Road, Shanghai 201203, China; State Key Laboratory of Genetic Engineering and MOE, Key Laboratory of Contemporary Anthropology, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China.
authors.forename: Wangluo
authors.initials: W
authors.lastname: Zhang
authors.forename: Shuai
authors.initials: S
authors.lastname: Chen
authors.forename: Yuanping
authors.initials: Y
authors.lastname: Liao
authors.forename: Dingli
authors.initials: D
authors.lastname: Wang
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Ding
authors.forename: Yingming
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Ran
authors.forename: Daru
authors.initials: D
authors.lastname: Lu
authors.forename: Huaxing
authors.initials: H
authors.lastname: Zhu
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent
keywords: Characterization; Coexpression; Formaldehyde dehydrogenase; Molecular chaperone; Protein purification; Pseudomonas aeruginosa