title: Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli.
abstract: In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M(r) approximately 55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca(2+)- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs.
authors.affiliation: Grup de Química Biològica i Biotecnologia, Institut Químic de Sarrià - Universitat Ramon Llull, Via Augusta 390, 08017, Barcelona, Spain.
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Carvajal-Vallejos
authors.forename: Alexandre
authors.initials: A
authors.lastname: Campos
authors.forename: Pablo
authors.initials: P
authors.lastname: Fuentes-Prior
authors.forename: Enrique
authors.initials: E
authors.lastname: Villalobos
authors.forename: André M
authors.initials: AM
authors.lastname: Almeida
authors.forename: Eduard
authors.initials: E
authors.lastname: Barberà
authors.forename: José María
authors.initials: JM
authors.lastname: Torné
authors.forename: Mireya
authors.initials: M
authors.lastname: Santos
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000378:metabolism*; D014508:Urea / Q000737:chemistry; D003313:Zea mays / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D014508:Urea; D011503:Transglutaminases; D019791:Guanidine