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 and Laurence BLANCHARD. Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies. Protein Expression and Purification. Elsevier, vol. 39, No 2, p. 296-306, 10 pp. ISSN 1046-5928. 2005.

Basic information

• Original name: Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies
• Name in Czech: Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from and optimized synthetic gene for NMR studies
• Authors: NÁLEZKOVÁ, Monika (203 Czech Republic, guarantor), Arjan DE GROOT (528 Netherlands), Marcus GRAF (276 Germany), Pierre GANS (250 France) and Laurence BLANCHARD (250 France).
• Edition: Protein Expression and Purification, Elsevier, 2005, 1046-5928.

Other information

• Original language: English
• Type of outcome: Article in a journal
• Field of Study: 10600 1.6 Biological sciences
• Country of publisher: France
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• Impact factor: Impact factor: 1.553
• Organization unit: Faculty of Science
• UT WoS: 000226437000021
• Keywords in English: PPAT; CoA biosynthesis; Optimized synthetic gene; Protein overexpression; NMR
• Tags: CoA biosynthesis, NMR, Optimized synthetic gene, PPAT, Protein overexpression

Abstract

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.

Abstract (in Czech)

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.