313
11. Acetone.
12. Cocktail for liquid scintillation counting such as Budget Solve
Complete Count Cocktail.
1. Microcentrifuge.
2. Heat block.
3. Cell incubator.
4. Cell hood.
5. Microscope for cell counting.
6. Scintillation counter.
3 Methods
1. Choose an appropriate site to split the kinase of interest. We have
utilized a dissimilarity based approach using sequence alignment
of several members of a particular kinase group. (see Note 1).
2. Choose desired CID system for your split kinase, either under
the control of rapamycin, abscisic acid, or gibberellic acid,
which heterodimerize the protein pairs FRB/FKBP, ABI cs
*
/
PYL cs , and GAI(92)/GID1, respectively.
3. Using standard cloning techniques (see Note 2), attach the
N-Terminal portion of the kinase (NTerm) to one of the CID
interacting proteins (CID1), and the C-Terminal portion of
the kinase (CTerm) to the other CID interacting protein
(CID2), to get the final NTerm-Linker-CID1 and CID2Linker-CTerm constructs (see Note 3). Make sure you use the
appropriate vector for your intended purpose (see Note 4).
4. Design one of your split kinase halves to include a His6-tag for
protein purification purposes. We commonly incorporate this
tag at the end of the kinase CTerm (CID2-Linker-CTermHis6) (see Note 5).
1. In order to use the in vitro rabbit reticulocyte lysate system,
in vitro translation (IVT) PCR linear products are generated
using the appropriate plasmid as templates, and using a forward primer that incorporates a RNA polymerase promoter,
such as T7, and a mammalian Kozak sequence, and a reverse
primer that incorporates a stabilizing RNA stem loop.
2. With the appropriate IVT PCR linear products in hand, generate mRNA products using a commercial RNA production system, such as Promega’s T7 RiboMAX Large Scale RNA
production system. Transcribe 3 μg of IVT-PCR products
using a RNA polymerase for 4 h at 30 °C (see Note 6) in a final
volume of 25 μL, with the provided reaction buffer and rNTPs.
mRNA is further purified using G50-microcolumns.
2.5 Instruments
and Equipment
3.1 Plasmid
Construction/Cloning
3.2 Split Kinase
Protein Expression
3.2.1 In Vitro Expression
in Rabbit Reticulocyte
Lysate
Split-Protein Kinases
11. Acetone.
12. Cocktail for liquid scintillation counting such as Budget Solve
Complete Count Cocktail.
1. Microcentrifuge.
2. Heat block.
3. Cell incubator.
4. Cell hood.
5. Microscope for cell counting.
6. Scintillation counter.
3 Methods
1. Choose an appropriate site to split the kinase of interest. We have
utilized a dissimilarity based approach using sequence alignment
of several members of a particular kinase group. (see Note 1).
2. Choose desired CID system for your split kinase, either under
the control of rapamycin, abscisic acid, or gibberellic acid,
which heterodimerize the protein pairs FRB/FKBP, ABI cs
*
/
PYL cs , and GAI(92)/GID1, respectively.
3. Using standard cloning techniques (see Note 2), attach the
N-Terminal portion of the kinase (NTerm) to one of the CID
interacting proteins (CID1), and the C-Terminal portion of
the kinase (CTerm) to the other CID interacting protein
(CID2), to get the final NTerm-Linker-CID1 and CID2Linker-CTerm constructs (see Note 3). Make sure you use the
appropriate vector for your intended purpose (see Note 4).
4. Design one of your split kinase halves to include a His6-tag for
protein purification purposes. We commonly incorporate this
tag at the end of the kinase CTerm (CID2-Linker-CTermHis6) (see Note 5).
1. In order to use the in vitro rabbit reticulocyte lysate system,
in vitro translation (IVT) PCR linear products are generated
using the appropriate plasmid as templates, and using a forward primer that incorporates a RNA polymerase promoter,
such as T7, and a mammalian Kozak sequence, and a reverse
primer that incorporates a stabilizing RNA stem loop.
2. With the appropriate IVT PCR linear products in hand, generate mRNA products using a commercial RNA production system, such as Promega’s T7 RiboMAX Large Scale RNA
production system. Transcribe 3 μg of IVT-PCR products
using a RNA polymerase for 4 h at 30 °C (see Note 6) in a final
volume of 25 μL, with the provided reaction buffer and rNTPs.
mRNA is further purified using G50-microcolumns.
2.5 Instruments
and Equipment
3.1 Plasmid
Construction/Cloning
3.2 Split Kinase
Protein Expression
3.2.1 In Vitro Expression
in Rabbit Reticulocyte
Lysate
Split-Protein Kinases