title: Engineered calcium-precipitable restriction enzyme.
abstract: We have developed a simple system for tagging and purifying proteins. Recent experiments have demonstrated that RTX (Repeat in Toxin) motifs from the adenylate cyclase toxin gene (CyaA) of B. pertussis undergo a conformational change upon binding calcium, resulting in precipitation of fused proteins and making this method a viable alternative for bioseparation. We have designed an iGEM Biobrick comprised of an RTX tag that can be easily fused to any protein of interest. In this paper, we detail the process of creating an RTX tagged version of the restriction enzyme EcoRI and describe a method for expression and purification of the functional enzyme.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, ∥BioFrontiers Institute, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Josephina
authors.initials: J
authors.lastname: Hendrix
authors.forename: Timothy
authors.initials: T
authors.lastname: Read
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Lalonde
authors.forename: Phillip K
authors.initials: PK
authors.lastname: Jensen
authors.forename: William
authors.initials: W
authors.lastname: Heymann
authors.forename: Elijah
authors.initials: E
authors.lastname: Lovelace
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Zimmermann
authors.forename: Michael
authors.initials: M
authors.lastname: Brasino
authors.forename: Joseph
authors.initials: J
authors.lastname: Rokicki
authors.forename: Robin D
authors.initials: RD
authors.lastname: Dowell
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D037361:Adenylate Cyclase Toxin; D011993:Recombinant Fusion Proteins; D015246:Deoxyribonuclease EcoRI; D002118:Calcium