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Purification and Characterization of Active Plk‐1 Produced in E. coli and Baculovirus‐Infected Sf9 Insect Cells
Author(s) -
Tam Amy Sze Pui,
Malakian Karl,
Benard Susan,
Kelleher Kerry,
Liu Yan,
Tan Xingzhi,
Loganzo Frank,
Siegel Marshall,
Kriz Ron,
Somers Will,
Stahl Mark,
Lin Laura
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb22
Subject(s) - polo like kinase , mitosis , biology , microbiology and biotechnology , cyclin dependent kinase 1 , kinase , protein kinase domain , sf9 , phosphorylation , cytokinesis , cell cycle , centrosome , cancer research , cell division , cell , biochemistry , mutant , recombinant dna , spodoptera , gene
The mammalian protein kinase Polo‐Like Kinase‐1 (Plk‐1) is an important regulator of mitosis. It is involved in the signaling networks that regulate many cell cycle processes such as centrosome maturation and separation, mitotic entry and exit, spindle formation and cytokinesis. Over‐expression of Plk‐1 was observed in a number of different tumors such as head and neck squamous cell carcinomas, ovarian and breast carcinomas and melanomas 1 . Cancer patients with over‐expressed Plk‐1 were predicted to have poor clinical outcome and shorten lifespan compared to those with moderate Plk‐1 expression 2 . Plk‐1 knockdown studies using anti‐Plk‐1 antibodies 3 or Plk‐1 siRNA 4 showed mitotic arrest and apoptosis. This validates Plk‐1 as a good candidate for small molecule inhibitor therapy to treat a wide range of cancer diseases. Human Plk‐1 consists of a N‐terminal Ser/Thr Kinase Domain (KD) and a regulatory C‐terminal Polo‐Box Domain (PBD). The PBD functions both as a Plk‐1 cellular localization domain as well as an inhibitory domain of the kinase. Upon binding to a phosphopeptide, such as Cdc25, PBD relinquishes the inhibitory effect thus allowing for phosphorylation and activation of the kinase domain. The active Plk‐1 then carries out the signaling cascade through phosphorylation of various downstream molecules such as Cdc2‐CyclinB complex and Anaphase Promoting Complex (APC) 5 . Presented here is the purification and characterization of Plk‐1 Full Length (expressed in Sf9 and E. coli cells) as well as Plk‐1 Kinase Domain (expressed in E. coli ).