2. Load the sample onto the column using a syringe and wash the
column with 8–10 CV of wash buffer.
3. Elute the protein with 5 CV wash buffer and collect 1.5 mL
fractions in 2 mL reaction tubes.
4. Measure the protein concentration of each fraction on a spectrophotometer at 280 nm. Then verify purity and molecular
weight of the protein with UPLC and LCMS, respectively.
Pool pure fractions to target a purity of >85–90% from UPLC.
5. Concentrate the combined sample using Amicon
® Ultra Centrifugal Filters with the suitable MWCO and measure the final
sample on LC-MS and UPLC.
3.4 Protein Thioester
Δ N PDZ Formation
and Isolation
1. Dialyze recombinant protein from pTWIN1-His 7 (Δ N PDZ)
against thiolysis buffer in a 1 L beaker. Replace dialysis buffer
with fresh thiolysis buffer every 2 h for at least 2 times. After
the last buffer exchange, allow the sample to dialyze overnight,
or about 8–12 h.
2. Transfer dialyzed protein sample into a 50 mL-conical tube and
add MESNa powder directly into the tube to target 100 mM
concentration. Adjust pH to 6.8 with 1 M HCl or NaOH, if
necessary.
3. Let thiolysis reaction proceed to form the protein thioester on a
rocking table at 4
C, for 0.5–3 days, as reaction time is highly
dependent on the intein and the amino acid at the cleavage
junction (see Note 8). Monitor reaction by removing 10 μL
aliquots every hour and analyze on LC-MS. Meanwhile, equilibrate a 5-mL His-trap FF column according to Subheading
3.3.2, step 3 with thiolysis buffer in preparation for protein
thioester purification.
4. Purify reaction mixture immediately upon completion (see
Note 9), by flowing the reaction mix through a His-trap FF
column, which sequesters the cleaved, histidine-tagged intein
and any uncleaved fusion protein. Collect eluent, which contains the protein thioester. Continue to elute with 5 CV thiolysis buffer and collect 1 CV per fraction.
5. Analyze fractions with LC-MS and pool the cleanest fractions.
If the final sample is below the target purity of 80% after this
step, the protein thioester can be further purified using SEC
(see Subheading 3.3.3).
6. Dialyze protein thioester against ultrapure water to remove
salts and buffer components. Freeze dry and lyophilize sample
to produce white solids. Characterize the final product with
UPLC and LC-MS. Store at À20
C until they are ready to be
used for processing.
Synthetic PDZ Domains
205
column with 8–10 CV of wash buffer.
3. Elute the protein with 5 CV wash buffer and collect 1.5 mL
fractions in 2 mL reaction tubes.
4. Measure the protein concentration of each fraction on a spectrophotometer at 280 nm. Then verify purity and molecular
weight of the protein with UPLC and LCMS, respectively.
Pool pure fractions to target a purity of >85–90% from UPLC.
5. Concentrate the combined sample using Amicon
® Ultra Centrifugal Filters with the suitable MWCO and measure the final
sample on LC-MS and UPLC.
3.4 Protein Thioester
Δ N PDZ Formation
and Isolation
1. Dialyze recombinant protein from pTWIN1-His 7 (Δ N PDZ)
against thiolysis buffer in a 1 L beaker. Replace dialysis buffer
with fresh thiolysis buffer every 2 h for at least 2 times. After
the last buffer exchange, allow the sample to dialyze overnight,
or about 8–12 h.
2. Transfer dialyzed protein sample into a 50 mL-conical tube and
add MESNa powder directly into the tube to target 100 mM
concentration. Adjust pH to 6.8 with 1 M HCl or NaOH, if
necessary.
3. Let thiolysis reaction proceed to form the protein thioester on a
rocking table at 4
C, for 0.5–3 days, as reaction time is highly
dependent on the intein and the amino acid at the cleavage
junction (see Note 8). Monitor reaction by removing 10 μL
aliquots every hour and analyze on LC-MS. Meanwhile, equilibrate a 5-mL His-trap FF column according to Subheading
3.3.2, step 3 with thiolysis buffer in preparation for protein
thioester purification.
4. Purify reaction mixture immediately upon completion (see
Note 9), by flowing the reaction mix through a His-trap FF
column, which sequesters the cleaved, histidine-tagged intein
and any uncleaved fusion protein. Collect eluent, which contains the protein thioester. Continue to elute with 5 CV thiolysis buffer and collect 1 CV per fraction.
5. Analyze fractions with LC-MS and pool the cleanest fractions.
If the final sample is below the target purity of 80% after this
step, the protein thioester can be further purified using SEC
(see Subheading 3.3.3).
6. Dialyze protein thioester against ultrapure water to remove
salts and buffer components. Freeze dry and lyophilize sample
to produce white solids. Characterize the final product with
UPLC and LC-MS. Store at À20
C until they are ready to be
used for processing.
Synthetic PDZ Domains
205