title: Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function.
abstract: High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Du
authors.forename: Pat
authors.initials: P
authors.lastname: Loulakis
authors.forename: Chun
authors.initials: C
authors.lastname: Luo
authors.forename: Anil
authors.initials: A
authors.lastname: Mistry
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Simons
authors.forename: Peter K
authors.initials: PK
authors.lastname: LeMotte
authors.forename: Francis
authors.initials: F
authors.lastname: Rajamohan
authors.forename: Kristina
authors.initials: K
authors.lastname: Rafidi
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Coleman
authors.forename: Kieran F
authors.initials: KF
authors.lastname: Geoghegan
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xie
mesh_terms: D000107:Acetylation; D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D064127:Aurora Kinases; D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000096:biosynthesis / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005942:Gluconates / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008027:Light; D008970:Molecular Weight; D019319:Okadaic Acid / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012542:Scattering, Radiation; D012694:Serine / Q000378:metabolism*; D018411:Spodoptera; D013917:Thrombin / Q000737:chemistry
chemical_list: D005942:Gluconates; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D019319:Okadaic Acid; D012694:Serine; D006639:Histidine; D051418:Focal Adhesion Kinase 2; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D000469:Alkaline Phosphatase; D013917:Thrombin; C030691:gluconic acid