2.9 Desulfurization
1. Solubilizing buffer: 0.2 M sodium phosphate buffer pH 7, 6 M
Gu·HCl.
2. Radical initiator VA-044.
3. Reduced glutathione.
4. 0.5 M TCEP stock solution in 500 μl 6 M Gu·HCl, 0.2 M
sodium phosphate pH 7.
2.10 Equipment
1. Mass Spectrometer with electron spray ionization (ESI) coupled to an HPLC system (ESI-LC/MS), equipped with a C18
reversed-phase column for the analysis of peptides and a C8
reversed-phase column for the characterization of proteins,
using a linear gradient of a binary buffer system containing
H 2 O/MeCN/TFA (A: 95/5/0.1; B: 5/95/0.1) at a flow
rate of 1 mL/min for proteins and H 2 O/MeCN/formic acid
(A: 95/5/0.1; B: 5/95/0.1) at a flow rate of 1 mL/min for
peptides.
2. Fast protein liquid chromatography (FPLC) system, equipped
with a gel filtration column for size-exclusion chromatography
(SEC), operating at a flow rate of 1 mL/min.
3. Cell disruptor system.
4. 4–15 L Freeze Dryer.
5. MiniBlock Shaker.
6. Benchtop pH Meter.
7. Analytical reversed-phase ultra-high performance liquid chromatography (RP-UPLC) system with a C18 reversed-phase
column for the analysis of peptides and a C8 reversed-phase
column for the analysis of proteins, using a binary buffer system
consisting of H 2 O/MeCN/TFA (A: 95/5/0.1; B: 5/95/0.1)
at 0.45 mL/min.
8. Preparative reversed-phase high-performance liquid chromatography (RP-HPLC) system with a C18 reversed-phase column for the purification of peptides and a reversed-phase C4
column for the purification of proteins, using a linear gradient
of a binary solvent system of H 2 O/MeCN/TFA (A: 95/5/
0.1; B: 5/95/0.1) at a flow rate of 20 mL/min.
3 Methods
3.1 Plasmid
Construction
for Expression
of Recombinant
Fragments
Prior to moving forward with the protocols below, a bacterial
expression vector with a gene encoding the protein-of-interest has
to be procured or cloned. Our PDZ domains are encoded in a
pRSET vector, so the following steps will refer to this plasmid,
pRSET(PDZ), but other constructs can be used as well.
Synthetic PDZ Domains
201
1. Solubilizing buffer: 0.2 M sodium phosphate buffer pH 7, 6 M
Gu·HCl.
2. Radical initiator VA-044.
3. Reduced glutathione.
4. 0.5 M TCEP stock solution in 500 μl 6 M Gu·HCl, 0.2 M
sodium phosphate pH 7.
2.10 Equipment
1. Mass Spectrometer with electron spray ionization (ESI) coupled to an HPLC system (ESI-LC/MS), equipped with a C18
reversed-phase column for the analysis of peptides and a C8
reversed-phase column for the characterization of proteins,
using a linear gradient of a binary buffer system containing
H 2 O/MeCN/TFA (A: 95/5/0.1; B: 5/95/0.1) at a flow
rate of 1 mL/min for proteins and H 2 O/MeCN/formic acid
(A: 95/5/0.1; B: 5/95/0.1) at a flow rate of 1 mL/min for
peptides.
2. Fast protein liquid chromatography (FPLC) system, equipped
with a gel filtration column for size-exclusion chromatography
(SEC), operating at a flow rate of 1 mL/min.
3. Cell disruptor system.
4. 4–15 L Freeze Dryer.
5. MiniBlock Shaker.
6. Benchtop pH Meter.
7. Analytical reversed-phase ultra-high performance liquid chromatography (RP-UPLC) system with a C18 reversed-phase
column for the analysis of peptides and a C8 reversed-phase
column for the analysis of proteins, using a binary buffer system
consisting of H 2 O/MeCN/TFA (A: 95/5/0.1; B: 5/95/0.1)
at 0.45 mL/min.
8. Preparative reversed-phase high-performance liquid chromatography (RP-HPLC) system with a C18 reversed-phase column for the purification of peptides and a reversed-phase C4
column for the purification of proteins, using a linear gradient
of a binary solvent system of H 2 O/MeCN/TFA (A: 95/5/
0.1; B: 5/95/0.1) at a flow rate of 20 mL/min.
3 Methods
3.1 Plasmid
Construction
for Expression
of Recombinant
Fragments
Prior to moving forward with the protocols below, a bacterial
expression vector with a gene encoding the protein-of-interest has
to be procured or cloned. Our PDZ domains are encoded in a
pRSET vector, so the following steps will refer to this plasmid,
pRSET(PDZ), but other constructs can be used as well.
Synthetic PDZ Domains
201