title: Co-expression of protein phosphatases in insect cells affects phosphorylation status and expression levels of proteins.
abstract: The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.
authors.affiliation: Faculty of Life Sciences, Manchester University, UK.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Brading
authors.forename: W Mark
authors.initials: WM
authors.lastname: Abbott
authors.forename: Isabelle
authors.initials: I
authors.lastname: Green
authors.forename: Alison
authors.initials: A
authors.lastname: Davies
authors.forename: Eileen J
authors.initials: EJ
authors.lastname: McCall
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005822:Genetic Vectors; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D036225:Receptor, EphB1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism* / Q000821:virology
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D036225:Receptor, EphB1; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases