3.1.1 Cloning of Plasmid
Encoding C-Terminal
Fragment
with N-Terminal Cys
For the generation of recombinant C-terminal fragment, the protein is expressed with a hexa-histidine purification tag and a recognition site for factor Xa, which upon cleavage after protein isolation
reveals the N-terminal Cys. The following protocol details the
cloning strategy, utilizing site-directed mutagenesis, to obtain the
plasmid encoding the C-fragment, pRSET(Δ C PDZ), starting with
the construct that encodes the full-length protein, pRSET(PDZ).
1. In a PCR tube, combine factor Xa primer pairs from Table 1,
the plasmid encoding full-length protein pRSET(PDZ), and
appropriate reagents, including a high-fidelity DNA polymerase from a site-directed mutagenesis kit.
2. Load PCR samples onto a thermocycler and execute temperature cycles according to manufacturer’s instructions.
3. To digest the DNA template, add 1 μL of DpnI restriction
enzyme to PCR mixture and let incubate at 37
C overnight, or
at least 8 h.
4. Transform 1 μL of PCR digest into 10 μL chemically competent TOP10 E. coli cells. After a heat-shock treatment of 45 s at
42
C followed by 2 min incubation on ice, grow cells in
250 μL of S.O.C medium at 37
C and 200 rpm for at least
45 min.
5. Transfer cells to sterile LB-ampicillin agar plates and incubate
overnight at 37
C.
6. Inoculate one colony into 5 mL of sterile LB-ampicillin media
and incubate overnight at 37
C and 200 rpm.
7. Isolate plasmid with DNA miniprep kit using manufacturer’s
instructions.
8. Confirm plasmid sequence with DNA sequencing to verify that
the mutations were inserted correctly into the plasmid and
store DNA at À20
C (see Note 3).
3.1.2 Gene Insertion into
an Intein-Encoding Plasmid
for the Generation
of Protein Thioesters
Below, is a protocol to prepare pTWIN1-His 7 (Δ N PDZ) by cloning
the PDZ domain fragment into a Mxe GyrA-encoding pTWIN1His 7 vector, which has been modified to contain a heptahistidine
tag. The original pTWIN1 vector with a chitin-binding domain
(CBD) that leverages the IMPACT protein purification system can
be used as well.
1. Using primer pairs from Table 1, amplify the gene encoding the
PDZ domain fragment with a high-fidelity DNA polymerase,
following manufacturer’s instructions for reagent amounts and
thermal cycles.
2. Digest PCR fragment and pTWIN1-His 7 vector with 1 μL each
of NdeI and SapI restriction enzymes at 37
C for 2 h. Isolate
digestion products with DNA gel electrophoresis. Extract fragment from agarose gel with DNA gel purification kit.
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Christin Kossmann et al.
Encoding C-Terminal
Fragment
with N-Terminal Cys
For the generation of recombinant C-terminal fragment, the protein is expressed with a hexa-histidine purification tag and a recognition site for factor Xa, which upon cleavage after protein isolation
reveals the N-terminal Cys. The following protocol details the
cloning strategy, utilizing site-directed mutagenesis, to obtain the
plasmid encoding the C-fragment, pRSET(Δ C PDZ), starting with
the construct that encodes the full-length protein, pRSET(PDZ).
1. In a PCR tube, combine factor Xa primer pairs from Table 1,
the plasmid encoding full-length protein pRSET(PDZ), and
appropriate reagents, including a high-fidelity DNA polymerase from a site-directed mutagenesis kit.
2. Load PCR samples onto a thermocycler and execute temperature cycles according to manufacturer’s instructions.
3. To digest the DNA template, add 1 μL of DpnI restriction
enzyme to PCR mixture and let incubate at 37
C overnight, or
at least 8 h.
4. Transform 1 μL of PCR digest into 10 μL chemically competent TOP10 E. coli cells. After a heat-shock treatment of 45 s at
42
C followed by 2 min incubation on ice, grow cells in
250 μL of S.O.C medium at 37
C and 200 rpm for at least
45 min.
5. Transfer cells to sterile LB-ampicillin agar plates and incubate
overnight at 37
C.
6. Inoculate one colony into 5 mL of sterile LB-ampicillin media
and incubate overnight at 37
C and 200 rpm.
7. Isolate plasmid with DNA miniprep kit using manufacturer’s
instructions.
8. Confirm plasmid sequence with DNA sequencing to verify that
the mutations were inserted correctly into the plasmid and
store DNA at À20
C (see Note 3).
3.1.2 Gene Insertion into
an Intein-Encoding Plasmid
for the Generation
of Protein Thioesters
Below, is a protocol to prepare pTWIN1-His 7 (Δ N PDZ) by cloning
the PDZ domain fragment into a Mxe GyrA-encoding pTWIN1His 7 vector, which has been modified to contain a heptahistidine
tag. The original pTWIN1 vector with a chitin-binding domain
(CBD) that leverages the IMPACT protein purification system can
be used as well.
1. Using primer pairs from Table 1, amplify the gene encoding the
PDZ domain fragment with a high-fidelity DNA polymerase,
following manufacturer’s instructions for reagent amounts and
thermal cycles.
2. Digest PCR fragment and pTWIN1-His 7 vector with 1 μL each
of NdeI and SapI restriction enzymes at 37
C for 2 h. Isolate
digestion products with DNA gel electrophoresis. Extract fragment from agarose gel with DNA gel purification kit.
202
Christin Kossmann et al.