title: Efficient and cost effective production of active-form human PKB using silkworm larvae.
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.
authors.affiliation: 1] Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan [2] Graduate School of Bioscience, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Maesaki
authors.affiliation: Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Satoh
authors.affiliation: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Taoka
authors.affiliation: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Kanaba
authors.affiliation: Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo, 192-0397, Japan.
authors.forename: Tsunaki
authors.initials: T
authors.lastname: Asano
authors.affiliation: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Fujita
authors.affiliation: Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan.
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Fujiwara
authors.affiliation: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Isobe
authors.affiliation: Graduate School of Bioscience, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Hakoshima
authors.affiliation: Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
authors.affiliation: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Mishima
mesh_terms: D000818:Animals; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007814:Larva / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D051057:Proto-Oncogene Proteins c-akt