3. We use T7-based expression vectors for PDZ domains, and
therefore T7 promoter forward (5- TAATACGACTCACTA
TAGGG -3) and the T7 terminator reverse (5- GCTAGT
TATTGCTCAGCGG -3) sequencing primers are typically
used to verify our plasmids.
4. Certain PDZ fragments when fused to an intein are expressed
as insoluble protein. Optimization of expression conditions
such as lowering temperature could allow expression of soluble
protein. Otherwise, it is possible to isolate and solubilize the
inclusion bodies with high concentration of denaturant (6 M
Gu·HCl or urea) prior to protein purification.
5. If the recombinant protein is expressed in inclusion bodies,
collect the protein pellet rather than the supernatant after
centrifugation. Then dissolve the pellet in lysis buffer supplemented with high concentrations of denaturant (e.g., 6 M
Gu·HCl). Incubate for 1 h until all particles are visually solubilized. Centrifuge the lysate at 50,000 Â g for 60 min and
collect supernatant. The lysate can then be purified using the
same IMAC steps but with elution buffers containing the same
concentration of denaturant in lysis buffer.
6. The pH should be adjusted to a pH above the pKa of His. pH 8
is attempted for enhancing the fraction of His being deprotonated and thus able to chelate to the Ni column.
7. The highest possible purity is desired to ensure a clean ligation
reaction with few site products and a better estimation of
equivalents used during NCL.
8. Reaction conditions may need to be optimized for different
proteins and inteins in order to maximize cleavage efficiency
while minimizing thioester hydrolysis. As a general rule, higher
thiolysis temperature, MESNa concentration, and reaction pH
would increase intein cleavage rate. However, if thioester
hydrolysis persists, adding urea to target a concentration
between 1–2 mM, as well as lowering reaction temperature
and pH can minimize the formation of the hydrolyzed side
product. For any pH adjustments performed during thiolysis, it
is also critical that the NaOH or HCl is added carefully so that
the reaction does not reach extreme ranges where the thioester
can hydrolyze.
9. If a pH adjustment is necessary before purification with a nickel
column, carefully apply 1 M NaOH or HCl to prevent the
sample from reaching extreme pH ranges, where the protein
thioester can hydrolyze.
10. Although performing the reaction at 4
C overnight, or
8–12 h, can prevent nonspecific cleavage of proteins, which is
known to occur in some PDZ domains, they can also result in
incomplete cleavage. To improve efficiency, more enzyme can
Synthetic PDZ Domains
213
therefore T7 promoter forward (5- TAATACGACTCACTA
TAGGG -3) and the T7 terminator reverse (5- GCTAGT
TATTGCTCAGCGG -3) sequencing primers are typically
used to verify our plasmids.
4. Certain PDZ fragments when fused to an intein are expressed
as insoluble protein. Optimization of expression conditions
such as lowering temperature could allow expression of soluble
protein. Otherwise, it is possible to isolate and solubilize the
inclusion bodies with high concentration of denaturant (6 M
Gu·HCl or urea) prior to protein purification.
5. If the recombinant protein is expressed in inclusion bodies,
collect the protein pellet rather than the supernatant after
centrifugation. Then dissolve the pellet in lysis buffer supplemented with high concentrations of denaturant (e.g., 6 M
Gu·HCl). Incubate for 1 h until all particles are visually solubilized. Centrifuge the lysate at 50,000 Â g for 60 min and
collect supernatant. The lysate can then be purified using the
same IMAC steps but with elution buffers containing the same
concentration of denaturant in lysis buffer.
6. The pH should be adjusted to a pH above the pKa of His. pH 8
is attempted for enhancing the fraction of His being deprotonated and thus able to chelate to the Ni column.
7. The highest possible purity is desired to ensure a clean ligation
reaction with few site products and a better estimation of
equivalents used during NCL.
8. Reaction conditions may need to be optimized for different
proteins and inteins in order to maximize cleavage efficiency
while minimizing thioester hydrolysis. As a general rule, higher
thiolysis temperature, MESNa concentration, and reaction pH
would increase intein cleavage rate. However, if thioester
hydrolysis persists, adding urea to target a concentration
between 1–2 mM, as well as lowering reaction temperature
and pH can minimize the formation of the hydrolyzed side
product. For any pH adjustments performed during thiolysis, it
is also critical that the NaOH or HCl is added carefully so that
the reaction does not reach extreme ranges where the thioester
can hydrolyze.
9. If a pH adjustment is necessary before purification with a nickel
column, carefully apply 1 M NaOH or HCl to prevent the
sample from reaching extreme pH ranges, where the protein
thioester can hydrolyze.
10. Although performing the reaction at 4
C overnight, or
8–12 h, can prevent nonspecific cleavage of proteins, which is
known to occur in some PDZ domains, they can also result in
incomplete cleavage. To improve efficiency, more enzyme can
Synthetic PDZ Domains
213