Isolation and characterization of human CapG expressed and post-translationally modified in Pichia pastoris.
CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.
Institute of Cell Biology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany. Electronic address: Agnes.Papala@hhu.de.
Agnes
A
Papala
Institute of Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany.
Marc
M
Sylvester
Heisenberg-Group - Environmentally-Induced Cardiovascular Degeneration, IUF - Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany.
Nadine
N
Dyballa-Rukes
Core Unit for Mass Spectrometry, IUF - Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany.
Sabine
S
Metzger
Institute of Cell Biology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.
Jochen
J
D'Haese
D000107:Acetylation; D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010766:Phosphorylation; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins
D008840:Microfilament Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C066575:CAPG protein, human
CapG; Heterologous expression; Mass-spectrometry; Pichia pastoris; Post-translational modifications; Tumor