Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum

Abstract

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.

Figure 1

Primary structure of the pea mitochondrial type II peroxiredoxin and alignment with its cytosolic counterpart from poplar. The signal peptide is coloured violet and the common residues are in yellow.

Figure 2

(a) Purification of mitochondrial type II Prx (lane 1, crude extract; lane 2, after Sephacryl S-200; lane 3, pure protein after MonoQ chromatography). (b) Peroxidase activity of the enzyme. (c) Crystal of recombinant mature pea mitochondrial type II peroxiredoxin.