Abstract：
:Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Maesaki R,Satoh R,Taoka M,Kanaba T,Asano T,Fujita C,Fujiwara T,Ito Y,Isobe T,Hakoshima T,Maenaka K,Mishima Mdoi
10.1038/srep06016subject
Has Abstractpub_date
2014-08-15 00:00:00pages
6016issn
2045-2322pii
srep06016journal_volume
4pub_type
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