Recombinant maize protoporphyrinogen IX oxidase expressed in Escherichia coli forms complexes with GroEL and DnaK chaperones.
The clone corresponding to maize plastidic protoporphyrinogen IX oxidase (PPO) has been isolated by functional complementation and inserted into a pET16b vector for expression in Escherichia coli. Recombinant PPO was purified by standard affinity chromatography using a metal chelating resin. Two contaminants copurified with recombinant PPO and were identified as GroEL and DnaK. Since chaperone binding to hydrophobic regions of the protein is regulated by ATP availability, an ATP washing step was introduced prior to elution of the recombinant protein from an affinity column. This washing step selectively removed both chaperones and allowed the recovery of pure PPO. Coexpression of PPO and GroELS resulted in a sixfold increase of soluble PPO yield, suggesting that bacterial chaperones could be limiting during the folding of the heterologous protein. However, a portion of PPO was still found in the insoluble fraction. Buffer containing the GroEL and DnaK enabled resuspension of PPO from the insoluble fraction but failed to enhance refolding of the denaturated protein. Attempts to increase the amount of soluble PPO using a thioredoxin-PPO fusion protein were not successful. Initial characterization of the recombinant PPO found that it possessed a high V(max), an elevated affinity for substrate, and an elevated sensitivity to PPO inhibitor herbicides compared to previous reports.
de Marco
A
A
LD Biochemistry Unit, Novartis Crop Protection AG, Basel, CH-4002, Switzerland.
Volrath
S
S
Bruyere
T
T
Law
M
M
Fonné-Pfister
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D018834:Chaperonin 60 / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
D018834:Chaperonin 60; D017931:DNA Primers; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; C063842:dnaK protein, E coli