Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.
Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824;
Dexin
D
Sui
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824;
Xinjing
X
Xu
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824;
Xuemei
X
Ye
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824;
Mengyu
M
Liu
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824;
Maxwell
M
Mianecki
¶Cell and Molecular Biology Program, Michigan State University, East Lansing, Michigan 48824.
Chotirat
C
Rattanasinchai
¶Cell and Molecular Biology Program, Michigan State University, East Lansing, Michigan 48824.
Christopher
C
Buehl
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824;
Xiexiong
X
Deng
From the ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824; kuom@msu.edu.
Min-Hao
MH
Kuo
D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015003:Yeasts; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C118136:TPPP protein, human; D016875:tau Proteins; D051360:Cyclin-Dependent Kinase 5; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3