Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from an optimized synthetic gene for NMR studies.
Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis in all organisms. This study was conducted to obtain a high amount of pure, soluble, and stable PPAT from the hyperthermophilic archaeon Pyrococcus abyssi with the aim of investigating its structural characterization by NMR. Production of this enzyme from its natural gene in the Escherichia coli classical expression strain (BL21(DE3)) was not possible, most likely due to the presence of a high number of E. coli rare codons. Only a low amount of P. abyssi PPAT was previously obtained in two E. coli strains encoding tRNAs that recognize these rare E. coli codons and only by using a very rich growth medium. It was not possible to use this strategy to prepare labelled samples for the NMR study, thus another solution had to be found. Therefore, a synthetic gene encoding P. abyssi PPAT was constructed for which not only the rare codons were changed but which was also optimized to avoid other expression-limiting factors such as internal ribosome entry sites, RNA secondary structures, and DNA repeats. Gene optimization strongly increased the yield of P. abyssi PPAT in E. coli BL21(DE3) and allowed us to start the structural characterization of the enzyme. Circular dichroism and 2D NMR experiments indicate the presence of a well-ordered structure for P. abyssi PPAT and also confirm the existence of this enzyme as a monomer in solution.
Nálezková
Monika
M
Laboratoire de Résonance Magnétique Nucléaire, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS-UJF), UMR 5075, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
de Groot
Arjan
A
Graf
Marcus
M
Gans
Pierre
P
Blanchard
Laurence
L
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D011522:Protons; D043903:Pyrococcus abyssi / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014170:Transformation, Genetic
D003062:Codon; D011522:Protons; D009713:Nucleotidyltransferases; C022315:pantetheine-phosphate adenylyltransferase