Limits...
Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: a cautionary tale.

Renz B, Davies JK, Carling D, Watkins H, Redwood C - Protein Expr. Purif. (2009)

Bottom Line: AMP-activated protein kinase (AMPK) is responsible for sensing of the cell's energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades.However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector.As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, University of Oxford, West Wing Level 6, John Radcliffe Hospital, Oxford OX3 9DU, UK.

ABSTRACT
AMP-activated protein kinase (AMPK) is responsible for sensing of the cell's energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the gamma 2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

Show MeSH

Related in: MedlinePlus

Phosphorylation of his6-C8 by AMPK. (A) One microgram of his6-C8 was phosphorylated by AMPK in the presence of γ32P-ATP as described in Materials and methods and separated by SDS–PAGE. Lane 1: Coomassie Blue-stained gel; lane 2: autoradiograph. (B) Time course of AMPK phosphorylation of 800 pmol SAMS (filled circles) and 800 pmol his6-C8 (open circles). Phosphorylation reactions were set up as described; aliquots were taken at the indicated time points and spotted onto phosphocellulose paper. The radioactivity remaining on the paper after washing with 10% TCA was determined by scintillation counting. (C) Phosphoamino acid analysis of 32P-labeled his6-C8. Total amino acids were separated by electrophoresis at pH 1.9 (first) and pH 3.5 and 32P-amino acids detected by autoradiography. Position of stained phosphoamino acid standards are marked.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691924&req=5

fig2: Phosphorylation of his6-C8 by AMPK. (A) One microgram of his6-C8 was phosphorylated by AMPK in the presence of γ32P-ATP as described in Materials and methods and separated by SDS–PAGE. Lane 1: Coomassie Blue-stained gel; lane 2: autoradiograph. (B) Time course of AMPK phosphorylation of 800 pmol SAMS (filled circles) and 800 pmol his6-C8 (open circles). Phosphorylation reactions were set up as described; aliquots were taken at the indicated time points and spotted onto phosphocellulose paper. The radioactivity remaining on the paper after washing with 10% TCA was determined by scintillation counting. (C) Phosphoamino acid analysis of 32P-labeled his6-C8. Total amino acids were separated by electrophoresis at pH 1.9 (first) and pH 3.5 and 32P-amino acids detected by autoradiography. Position of stained phosphoamino acid standards are marked.

Mentions: pET28a-C8 DNA was used to transform BL21(DE3)pLysS Escherichia coli and his6-C8 protein was overexpressed in cultures of transformed cells grown in L broth containing 30 μg/ml kanamycin and 25 μg/ml chloramphenicol at 37 °C. Expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to 0.4 mM after which the cultures were grown for a further 3 h; cells were harvested by centrifugation and the cell pellets frozen at −80 °C. For purification, cell pellets were thawed, resuspended in 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10 mM imidazole and cell lysis brought about by sonication. Cell debris was removed by centrifugation (20,000 g for 20 min at 4 °C) and his6-C8 protein isolated from the lysis supernatant using Ni2+–NTA (Qiagen) affinity chromatography according to the manufacturer’s instructions (Fig. 2A). Digestion of his6-C8 fusion protein with thrombin (1 U/μg protein) was carried out at 4 °C overnight in 20 mM Tris–HCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl2.


Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: a cautionary tale.

Renz B, Davies JK, Carling D, Watkins H, Redwood C - Protein Expr. Purif. (2009)

Phosphorylation of his6-C8 by AMPK. (A) One microgram of his6-C8 was phosphorylated by AMPK in the presence of γ32P-ATP as described in Materials and methods and separated by SDS–PAGE. Lane 1: Coomassie Blue-stained gel; lane 2: autoradiograph. (B) Time course of AMPK phosphorylation of 800 pmol SAMS (filled circles) and 800 pmol his6-C8 (open circles). Phosphorylation reactions were set up as described; aliquots were taken at the indicated time points and spotted onto phosphocellulose paper. The radioactivity remaining on the paper after washing with 10% TCA was determined by scintillation counting. (C) Phosphoamino acid analysis of 32P-labeled his6-C8. Total amino acids were separated by electrophoresis at pH 1.9 (first) and pH 3.5 and 32P-amino acids detected by autoradiography. Position of stained phosphoamino acid standards are marked.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691924&req=5

fig2: Phosphorylation of his6-C8 by AMPK. (A) One microgram of his6-C8 was phosphorylated by AMPK in the presence of γ32P-ATP as described in Materials and methods and separated by SDS–PAGE. Lane 1: Coomassie Blue-stained gel; lane 2: autoradiograph. (B) Time course of AMPK phosphorylation of 800 pmol SAMS (filled circles) and 800 pmol his6-C8 (open circles). Phosphorylation reactions were set up as described; aliquots were taken at the indicated time points and spotted onto phosphocellulose paper. The radioactivity remaining on the paper after washing with 10% TCA was determined by scintillation counting. (C) Phosphoamino acid analysis of 32P-labeled his6-C8. Total amino acids were separated by electrophoresis at pH 1.9 (first) and pH 3.5 and 32P-amino acids detected by autoradiography. Position of stained phosphoamino acid standards are marked.
Mentions: pET28a-C8 DNA was used to transform BL21(DE3)pLysS Escherichia coli and his6-C8 protein was overexpressed in cultures of transformed cells grown in L broth containing 30 μg/ml kanamycin and 25 μg/ml chloramphenicol at 37 °C. Expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to 0.4 mM after which the cultures were grown for a further 3 h; cells were harvested by centrifugation and the cell pellets frozen at −80 °C. For purification, cell pellets were thawed, resuspended in 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10 mM imidazole and cell lysis brought about by sonication. Cell debris was removed by centrifugation (20,000 g for 20 min at 4 °C) and his6-C8 protein isolated from the lysis supernatant using Ni2+–NTA (Qiagen) affinity chromatography according to the manufacturer’s instructions (Fig. 2A). Digestion of his6-C8 fusion protein with thrombin (1 U/μg protein) was carried out at 4 °C overnight in 20 mM Tris–HCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl2.

Bottom Line: AMP-activated protein kinase (AMPK) is responsible for sensing of the cell's energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades.However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector.As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, University of Oxford, West Wing Level 6, John Radcliffe Hospital, Oxford OX3 9DU, UK.

ABSTRACT
AMP-activated protein kinase (AMPK) is responsible for sensing of the cell's energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the gamma 2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

Show MeSH
Related in: MedlinePlus