title: Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA.
abstract: Five independent clones of the Propionibacterium acnes P-37 lipase gene (gehA) were obtained in Escherichia coli, and the gene was localized to a 2.75 kb Xhol fragment by subcloning. The five clones were shown to contain the same gene by Southern blotting with a DIG-labelled probe to gehA. The nucleotide sequence of gehA was determined, and shown to contain a single ORF of 1017 kb, encoding a protein of 339 amino acids. The predicted molecular mass was 36 kDa. A 33 kDa (PAGE) radiolabelled polypeptide was detected from E. coli minicell preparations harbouring gehA, which could correspond to GehA after cleavage of the putative 26 amino acid residue signal peptide. gehA was overexpressed in E. coli under the control of the bacteriophage T7 promoter, and the corresponding polypeptide was found to be present in insoluble aggregates. Active lipase was produced when the overexpressing strain was incubated at a reduced temperature in the presence of sucrose. Purification of lipase from P. acnes culture supernatant fluids confirmed the production of a 33 kDa (PAGE) lipase.
authors.affiliation: Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 ONF, UK.
authors.forename: James E
authors.initials: JE
authors.lastname: Miskin
authors.affiliation: Skin Research Centre, Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Farrell
authors.affiliation: Leeds Foundation for Dermatological Research, Department of Dermatology, The General Infirmary at Leeds, Great George Street, Leeds LS1 3EX, UK.
authors.forename: William J
authors.initials: WJ
authors.lastname: Cunliffe
authors.affiliation: Skin Research Centre, Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Keith T
authors.initials: KT
authors.lastname: Holland
mesh_terms: D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008049:Lipase / Q000235:genetics*; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011425:Propionibacterium acnes / Q000201:enzymology* / Q000235:genetics; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D008049:Lipase