316
5. Compare your activated split kinase samples (+CID), nonactivated ones (−CID), as well as the negative control, for
example, RRL or cell lysate only, to confirm that the split
kinase is small molecule dependent.
4 Notes
1. We have found three general fragmentation sites for tyrosine
kinases, equivalent to Lyn kinase 298/299, 311/312, and
393/394 sites (using the numbering system in UniProt
P07948) [25]. These are a good starting point to create a
working split kinase system for protein tyrosine kinases.
2. In brief, a standard cloning procedure consists of generating
desired inserts by PCR amplification using appropriate oligonucleotide primers, followed by digestion with appropriate
restriction enzymes. A selected vector is digested using the
same restriction enzymes, and the insert and vector are
subsequently ligated using a DNA ligase. The ligated product
is then used to transform competent cells, grow colonies,
purify plasmid DNA in those colonies, and finally confirm the
intended construct by DNA sequencing.
3. We most commonly use the NTerm-CID1 and CID2-CTerm
arrangement, but different arrangements and orientations should
be tried to optimize a particular split kinase system. The most
practical way to generate these constructs is by creating a vector
with two multiple cloning sites (MCS) separated by a linker
(MCS-Linker-MCS). The CID interacting proteins can then be
cloned to generate general CID vectors, such as MCS-LinkerCID1 and CID2-Linker-MCS. We have used different linker
lengths (13, 17, 21 and 25aa, with GGGS repeats), and several
should be tried to optimize a particular split kinase system.
4. We most commonly use the pRSFDuet-1 vector from Novagen
and the pcDNA3.1(+) vector from Invitrogen. Both of them
can be used to produce protein using in vitro methods, while
only the pcDNA3.1(+) vector is used to express proteins in
mammalian cells.
5. We have previously placed the His6-tag at the N-termini of the
constructs, with equally successful results. Other purification
tags, such as FLAG-tag, can also be used in place of the
His6-tag.
6. If mRNA yields are low at 4 h and 30 °C, yields may improve
with 4 h at 37 °C.
7. A ribonuclease inhibitor, such as RNasin, can be added to the
RLL reaction to prevent mRNA degradation. If the RLL sysJavier Castillo-Montoya and Indraneel Ghosh
5. Compare your activated split kinase samples (+CID), nonactivated ones (−CID), as well as the negative control, for
example, RRL or cell lysate only, to confirm that the split
kinase is small molecule dependent.
4 Notes
1. We have found three general fragmentation sites for tyrosine
kinases, equivalent to Lyn kinase 298/299, 311/312, and
393/394 sites (using the numbering system in UniProt
P07948) [25]. These are a good starting point to create a
working split kinase system for protein tyrosine kinases.
2. In brief, a standard cloning procedure consists of generating
desired inserts by PCR amplification using appropriate oligonucleotide primers, followed by digestion with appropriate
restriction enzymes. A selected vector is digested using the
same restriction enzymes, and the insert and vector are
subsequently ligated using a DNA ligase. The ligated product
is then used to transform competent cells, grow colonies,
purify plasmid DNA in those colonies, and finally confirm the
intended construct by DNA sequencing.
3. We most commonly use the NTerm-CID1 and CID2-CTerm
arrangement, but different arrangements and orientations should
be tried to optimize a particular split kinase system. The most
practical way to generate these constructs is by creating a vector
with two multiple cloning sites (MCS) separated by a linker
(MCS-Linker-MCS). The CID interacting proteins can then be
cloned to generate general CID vectors, such as MCS-LinkerCID1 and CID2-Linker-MCS. We have used different linker
lengths (13, 17, 21 and 25aa, with GGGS repeats), and several
should be tried to optimize a particular split kinase system.
4. We most commonly use the pRSFDuet-1 vector from Novagen
and the pcDNA3.1(+) vector from Invitrogen. Both of them
can be used to produce protein using in vitro methods, while
only the pcDNA3.1(+) vector is used to express proteins in
mammalian cells.
5. We have previously placed the His6-tag at the N-termini of the
constructs, with equally successful results. Other purification
tags, such as FLAG-tag, can also be used in place of the
His6-tag.
6. If mRNA yields are low at 4 h and 30 °C, yields may improve
with 4 h at 37 °C.
7. A ribonuclease inhibitor, such as RNasin, can be added to the
RLL reaction to prevent mRNA degradation. If the RLL sysJavier Castillo-Montoya and Indraneel Ghosh