Electron paramagnetic resonance as a tool for monitoring overexpression in Escherichia coli of fully functional flavodoxin.
Overexpression of foreign proteins in Escherichia coli is usually tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Western blot analysis. However, when metalloproteins of flavoenzymes are expressed, the correct assembly of the polypeptide chain to the cofactor cannot be verified using these methods. We have used EPR spectroscopy and Mancini tests to monitor the expression of holoflavodoxin from the cyanobacteria Anabaena in E. coli. Flavodoxin from Anabaena sp PCC 7119 was cloned in the plasmid pTrc 99b after the trc promotor, which is activated in the presence of isopropyl-beta-D-thiogalactoside. Two hours after induction, most of the recombinant apoflavodoxin is already synthesized. However, only 65% of this protein is assembled to flavin mononucleotide (FMN). Harvesting of the cells to obtain all the flavodoxin in the holo form must be carried out 10 h after induction. Addition of FMN to the culture media increases the synthesis of holoflavodoxin by about 17%. The presence of flavin adenine dinucleotide or riboflavin in the culture inhibits the accumulation of semiquinone flavodoxin in the cells.
Borruel
A
A
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.
Peleato
M L
ML
Orera
V M
VM
Gómez-Moreno
C
C
Fillat
M F
MF
D001059:Apoproteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005418:Flavodoxin / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D001059:Apoproteins; D005418:Flavodoxin; D011994:Recombinant Proteins