Journal article
Large-scale expression and purification of a soluble form of the pleckstrin homology domain of the human protooncogenic serine/threonine protein kinase PKB (c-Akt) in Escherichia coli
Protein Expression and Purification, Vol.17(2), pp.224-230
1999
Abstract
The protooncogenic serine/threonine protein kinase PKB contains an amino-terminal pleckstrin homology (PH) domain which binds phosphatidylinositides. The PH domain, composed of ~O100 loosely conserved amino acids, is found in many proteins, including kinases, phospholipases C, GTPases, GTPase-activating proteins, GTPase-exchange factors, 'adaptor' proteins, cytoskeletal proteins, and kinase substrates. We have developed an expression system in Escherichia coli that can produce large quantities of a soluble form of the PKB PH domain and have purified it to apparent homogeneity. Expression of the PKB PH domain as a (His)6-tagged fusion with the addition of 3 lysines at the carboxyl-terminus facilitated the production of soluble protein. Induction of expression at 24°C as opposed to 37°C also significantly increased solubility of the PH domain. Large-scale purification was easily achieved by exploiting the (His)6 tag and the high isoelectric point of the protein attributable to the additional 3 carboxyl-terminal lysines.
Details
- Title
- Large-scale expression and purification of a soluble form of the pleckstrin homology domain of the human protooncogenic serine/threonine protein kinase PKB (c-Akt) in Escherichia coli
- Authors/Creators
- E. Ingley (Author/Creator)B.A. Hemmings (Author/Creator)
- Publication Details
- Protein Expression and Purification, Vol.17(2), pp.224-230
- Publisher
- Academic Press
- Identifiers
- 991005542558507891
- Copyright
- © 1999 Academic Press.
- Murdoch Affiliation
- Murdoch University
- Language
- English
- Resource Type
- Journal article
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- 1.96 Cell Biology
- 1.96.302 Membrane Trafficking
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