Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies

NÁLEZKOVÁ, Monika, Arjan DE GROOT, Marcus GRAF, Pierre GANS a Laurence BLANCHARD. Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies. Online. Protein Expression and Purification. Elsevier, 2005, roč. 39, č. 2, s. 296-306, 10 s. ISSN 1046-5928. [citováno 2024-04-23]

Základní údaje

• Originální název: Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies
• Název česky: Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies
• Autoři: NÁLEZKOVÁ, Monika (203 Česká republika, garant), Arjan DE GROOT (528 Nizozemské království), Marcus GRAF (276 Německo), Pierre GANS (250 Francie) a Laurence BLANCHARD (250 Francie)
• Vydání: Protein Expression and Purification, Elsevier, 2005, 1046-5928.

Další údaje

• Originální jazyk: angličtina
• Typ výsledku: Článek v odborném periodiku
• Obor: 10600 1.6 Biological sciences
• Stát vydavatele: Francie
• Utajení: není předmětem státního či obchodního tajemství
• WWW: URL
• Impakt faktor: Impact factor: 1.553
• Organizační jednotka: Přírodovědecká fakulta
• UT WoS: 000226437000021
• Klíčová slova anglicky: PPAT; CoA biosynthesis; Optimized synthetic gene; Protein overexpression; NMR
• Štítky: CoA biosynthesis, NMR, Optimized synthetic gene, PPAT, Protein overexpression

Anotace

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.

Anotace česky

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.