title: Analysis of conditions affecting auto-phosphorylation of human kinases during expression in bacteria.
abstract: Bacterial over-expression of kinases is often associated with high levels of auto-phosphorylation resulting in heterogeneous recombinant protein preparations or sometimes in insoluble protein. Here we present expression systems for nine kinases in Escherichia coli and, for the most heavily phosphorylated, the characterisation of factors affecting auto-phosphorylation. Experiments showed that the level of auto-phosphorylation was proportional to the rate of expression. Comparison of phosphorylation states following in vitro phosphorylation with phosphorylation states following expression in E. coli showed that the non-physiological 'hyper-phosphorylation' was occurring at sites that would require local unfolding to be accessible to a kinase active site. In contrast, auto-phosphorylation on unphosphorylated kinases that had been expressed in bacteria overexpressing λ-phosphatase was only observed on distinct exposed sites. Remarkably, the Ser/Thr kinase PLK4 auto-phosphorylated on a tyrosine residue (Tyr177) located in the activation segment. The results give support to a mechanism in which auto-phosphorylation occurs before or during protein folding. In addition, the expression systems and protocols presented will be a valuable resource to the research community.
authors: Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors: Amit
authors: A
authors: Shrestha
authors: Gray Institute for Radiation Oncology and Biology, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors: Garth
authors: G
authors: Hamilton
authors: Gray Institute for Radiation Oncology and Biology, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors: Eric
authors: E
authors: O'Neill
authors: Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors: Stefan
authors: S
authors: Knapp
authors: Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK. Electronic address: jon.elkins@sgc.ox.ac.uk.
authors: Jonathan M
authors: JM
authors: Elkins
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases