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.
The protooncogenic serine/threonine protein kinase PKB contains an amino-terminal pleckstrin homology (PH) domain which binds phosphatidylinositides. The PH domain, composed of approximately 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.
Ingley
E
E
Friedrich Miescher-Institut, Basel, CH-4002, Switzerland.
Hemmings
B A
BA
D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D051057:Proto-Oncogene Proteins c-akt; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014169:Transformation, Bacterial
D011518:Proto-Oncogene Proteins; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt