title: Chaperone over-expression in Escherichia coli: apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates.
abstract: The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show approximately 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.
authors.affiliation: Novartis Institutes for BioMedical Research, Center for Proteomic Chemistry, Novartis Campus, CH-4002 Basel, Switzerland. annette.haacke@novartis.com
authors.forename: Annette
authors.initials: A
authors.lastname: Haacke
authors.forename: Gabriele
authors.initials: G
authors.lastname: Fendrich
authors.forename: Paul
authors.initials: P
authors.lastname: Ramage
authors.forename: Martin
authors.initials: M
authors.lastname: Geiser
mesh_terms: D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D018832:Molecular Chaperones; D011994:Recombinant Proteins; D010770:Phosphotransferases