Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum.
A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 angstroms from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 angstroms, alpha = 102.90, beta = 104.40, gamma = 99.07 degrees, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.
Barranco-Medina
Sergio
S
Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada, Spain.
López-Jaramillo
Francisco Javier
FJ
Bernier-Villamor
Laura
L
Sevilla
Francisca
F
Lázaro
Juan José
JJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D018076:DNA, Complementary; D004926:Escherichia coli; D008928:Mitochondria / Q000737:chemistry; D016366:Open Reading Frames; D018532:Pisum sativum; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D054464:Peroxiredoxins; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D054464:Peroxiredoxins