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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

Ingley, E.ORCID: 0000-0002-8112-9134 and Hemmings, B.A. (1999) 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, 17 (2). pp. 224-230.

Link to Published Version: https://doi.org/10.1006/prep.1999.1120
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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.

Item Type: Journal Article
Publisher: Academic Press
Copyright: © 1999 Academic Press.
URI: http://researchrepository.murdoch.edu.au/id/eprint/39584
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