title: Efficient and Flexible Preparation of Biosynthetic Microperoxidases.
abstract: Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as N-terminal tags to the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., and Bren, K. L. (2010) Protein Sci. 19, 1830-1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides.
authors.affiliation: Department of Chemistry, University of Rochester , Rochester, New York 14627-0216, United States.
authors.forename: Erin C
authors.initials: EC
authors.lastname: Kleingardner
authors.affiliation: Department of Chemistry, University of Rochester , Rochester, New York 14627-0216, United States.
authors.forename: Wesley B
authors.initials: WB
authors.lastname: Asher
authors.affiliation: Department of Chemistry, University of Rochester , Rochester, New York 14627-0216, United States.
authors.forename: Kara L
authors.identifier: http://orcid.org/0000-0002-8082-3634
authors.initials: KL
authors.lastname: Bren
mesh_terms: D000595:Amino Acid Sequence; D001400:Azurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010544:Peroxidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001427:Bacterial Toxins; D010455:Peptides; D011994:Recombinant Proteins; C434464:RtxA protein, Vibrio cholerae; D001400:Azurin; D006418:Heme; D010544:Peroxidases; C021799:microperoxidase