Browse views: by Year, by Function, by GLF, by Subfunction, by Conference, by Journal

Chaperone over-expression in Escherichia coli: apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates.

Haacke, Annette and Fendrich, Gabriele and Ramage, Paul and Geiser, Martin (2009) Chaperone over-expression in Escherichia coli: apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates. Protein Expression and Purification, 64 (2). pp. 185-193. ISSN 1096-0279

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.

Item Type: Article
Related URLs:
Additional Information: Author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords: Protein kinases; Escherichia coli; Molecular chaperone; Protein folding; Co-expression; Protein purification
Related URLs:
Date Deposited: 22 Feb 2010 11:50
Last Modified: 31 Jan 2013 00:50
URI: https://oak.novartis.com/id/eprint/1769

Search

Email Alerts

Register with OAK to receive email alerts for saved searches.