3. Add the C-terminal peptide to the reaction mixture and adjust
pH carefully to 6.8–7.0 with NaOH or HCl (see Note 29).
4. Incubate the reaction for 2–8 h at 25
C on a shaker at
500 rpm. If overnight reactions are necessary, incubate the
solution at 4
C while constant shaking.
5. Quench the reaction by adding 60 eq TCEP from the TCEP
stock solution.
6. The ligation product can be purified directly, either via sizeexclusion chromatography (see Subheading 3.3.3) or via
preparative HPLC.
3.10 Desulfurization
Since the native chemical ligation reaction often requires the mutation of Ala to Cys, a conversion of the Cys to Ala might be necessary
to remove EPL artifacts. This step can be performed by metal-free
desulfurization with TCEP, a radical initiator (VA-044) and
reduced glutathione.
1. Place the ligation product in a 1.5 mL reaction vessel and
dissolve it in solubilizing buffer to a final concentration of
2.5 mM.
2. Add 0.5 M TCEP solution to the peptide to achieve a final
TCEP concentration of 0.2 M. Mix by vortexing.
3. Add reduced glutathione (solid) to a final concentration of
0.04 M.
4. Add VA-044 (solid) to a final concentration of 0.02 M.
5. The reaction takes 5–20 h at RT, approximately 20
C, on a
shaker at 500 rpm (see Note 30).
6. Test the completion of desulfurization periodically by removing 10 μL sample and measure it by LC-MS and UPLC.
4 Notes
1. Procuring or constructing a plasmid that encodes the fulllength protein-of-interest will give flexibility in the cloning of
all recombinant fragments for EPL. For PDZ domains, we
purchased the full-length sequence of the PDZ domain and
cloned it into pRSET vector. This construct can be used either
in mutagenesis to generate a plasmid encoding C-fragment, or
as the DNA template in PCR to generate the fragment that
would be cloned into an intein-encoding plasmid.
2. Six (Ala, Gly, Phe, Ile, Leu, Met, Asn, Gln, and Val) of the
nineteen α-hydroxy acids are commercially available.
212
Christin Kossmann et al.
pH carefully to 6.8–7.0 with NaOH or HCl (see Note 29).
4. Incubate the reaction for 2–8 h at 25
C on a shaker at
500 rpm. If overnight reactions are necessary, incubate the
solution at 4
C while constant shaking.
5. Quench the reaction by adding 60 eq TCEP from the TCEP
stock solution.
6. The ligation product can be purified directly, either via sizeexclusion chromatography (see Subheading 3.3.3) or via
preparative HPLC.
3.10 Desulfurization
Since the native chemical ligation reaction often requires the mutation of Ala to Cys, a conversion of the Cys to Ala might be necessary
to remove EPL artifacts. This step can be performed by metal-free
desulfurization with TCEP, a radical initiator (VA-044) and
reduced glutathione.
1. Place the ligation product in a 1.5 mL reaction vessel and
dissolve it in solubilizing buffer to a final concentration of
2.5 mM.
2. Add 0.5 M TCEP solution to the peptide to achieve a final
TCEP concentration of 0.2 M. Mix by vortexing.
3. Add reduced glutathione (solid) to a final concentration of
0.04 M.
4. Add VA-044 (solid) to a final concentration of 0.02 M.
5. The reaction takes 5–20 h at RT, approximately 20
C, on a
shaker at 500 rpm (see Note 30).
6. Test the completion of desulfurization periodically by removing 10 μL sample and measure it by LC-MS and UPLC.
4 Notes
1. Procuring or constructing a plasmid that encodes the fulllength protein-of-interest will give flexibility in the cloning of
all recombinant fragments for EPL. For PDZ domains, we
purchased the full-length sequence of the PDZ domain and
cloned it into pRSET vector. This construct can be used either
in mutagenesis to generate a plasmid encoding C-fragment, or
as the DNA template in PCR to generate the fragment that
would be cloned into an intein-encoding plasmid.
2. Six (Ala, Gly, Phe, Ile, Leu, Met, Asn, Gln, and Val) of the
nineteen α-hydroxy acids are commercially available.
212
Christin Kossmann et al.