title: Expression and structure/function relationships of human defensin 5.
abstract: The innate immunity utilizes a battery of broad-spectrum antibacterial cationic polypeptides (3-5 kDa), among them defensins. In humans, defensins are the first line of defense against pathogens and their expression has been implicated in several diseases. The antibacterial activity of defensins is generally ascribed to their overall positive charge, which enables them to disrupt bacterial membrane integrity and function, but their active surface has not been fully elucidated. To perform structural and functional assays, an efficient, high-yield, easy-to-use expression and purification system must be established. Up to now, most efforts to obtain larger quantities of active recombinant defensins have been only moderately successful. Herein, we report the establishment of an efficient, high-yield expression and purification system for human defensin 5 (HD-5). Using site-directed mutagenesis, we pinpoint several arginine residues and Y27 as important for HD-5 antibacterial activity. Our expression and purification system can be harnessed for structure/activity relationship studies of defensins in particular and small polypeptides in general.
authors.affiliation: Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
authors.forename: Nava
authors.initials: N
authors.lastname: Chapnik
authors.forename: Anat
authors.initials: A
authors.lastname: Levit
authors.forename: Masha Y
authors.initials: MY
authors.lastname: Niv
authors.forename: Oren
authors.initials: O
authors.lastname: Froy
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D001120:Arginine / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D055672:Static Electricity; D013329:Structure-Activity Relationship; D023084:alpha-Defensins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; C500225:DEFA5 protein, human; D011994:Recombinant Proteins; D023084:alpha-Defensins; D001120:Arginine