pETPhos: a customized expression vector designed for further characterization of Ser/Thr/Tyr protein kinases and their substrates.
Bacterial genomics revealed the widespread distribution of serine/threonine protein kinases (STPKs), which regulate various cellular processes. However, understanding the role of phosphorylation in prokaryotes has been hampered by the paucity of endogenous substrates identified and the restricted number of tools allowing identification and characterization of the phosphoresidues. Herein, we describe an improved vector, pETPhos, to express proteins harboring a N-terminal His-tag fusion, which can be efficiently removed using the TEV protease. One major advantage of pETPhos relies on the lack of Ser and Thr residues in the fusion tag, representing potential non-specific phosphorylation sites. The usefulness of pETPhos is illustrated by a comparative analysis in which the Mycobacterium tuberculosis protein Rv2175c, a substrate of the STPK PknL, is expressed either in a pET28 derivative or in pETPhos. Following in vitro phosphorylation with PknL, phosphoaminoacid analysis revealed the presence of phosphorylated Ser and Thr in Rv2175c expressed in the pET28 derivative. However, when expressed in pETPhos, only Thr were phosphorylated. These findings indicate that STPKs can phosphorylate Ser-containing His-tag fusions, thus conducting to misleading results. We demonstrate that pETPhos represents a valuable tool for characterization of the phosphoacceptors in bacterial STPKs, and presumably also in Tyr protein kinases, as well as in their substrates.
Canova
Marc J
MJ
Institut de Biologie et Chimie des Protéines (IBCP UMR 5086), CNRS, Université Lyon1, IFR128 BioSciences, Lyon-Gerland, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
Kremer
Laurent
L
Molle
Virginie
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases