The protooncogenic serine/threonine protein kinase PKB contains an amino-terminal pleckstrin homology (PH) domain which binds phosphatidylinositides. The PH domain, composed of similar to 100 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 degrees C as opposed to 37 degrees 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. (C) 1999 Academic Press.