3.5 Factor Xa
Cleavage to Generate
N-Terminal Cys
Protein Fragment
Δ C PDZ
1. Dialyze recombinant protein from pRSET(Δ C PDZ) against
1 L of factor Xa buffer at 4
C. Exchange dialysis buffer with
fresh buffer every 2 h for 3 times. Allow sample to dialyze
overnight after the last buffer exchange.
2. Transfer protein sample to a fresh 50 mL-conical tube. Add
factor Xa according to manufacturer’s instructions and allow
enzymatic cleavage to progress at 4
C on a rotating or rocking
table for 12–24 h. Remove 10 μL aliquots every hour and
monitor reaction with LC-MS (see Note 10).
3. Prepare a 5-mL His-trap FF column for purification by following Subheading 3.3.2, step 3 with cleavage buffer. Purify
cleavage mixture immediately after reaction is complete to
avoid nonspecific cleavage, using steps 4 and 5 in Subheading
3.4 with factor Xa cleavage buffer as the eluent.
4. Dialyze the final protein fragment against ultrapure water to
remove salt and buffer components, and lyophilize protein to
form white amorphous solids. Analyze compound with UPLC
and LC-MS, and store at À20
C until they are ready to be used
for processing.
3.6 Solid-Phase
Peptide Synthesis
SPPS can be performed with two protection techniques: Boc/Bzl
or Fmoc/t-Bu. The latter has the advantage of an orthogonal
protection system, which allows milder conditions and a broader
range in the pH-dependent reaction conditions for selective cleavage of side-chain protection groups and the Fmoc-group [3]. The
Fmoc-SPPS method is preferred over Boc-SPPS in regard to phosphorylated peptides because phosphate groups are not stable during HF cleavage. Several automated synthesizers are available,
enabling fast and convenient SPPS. For the inclusion of expensive
building blocks, such as phospho-amino acids, manual synthesis is
often preferred, because less material is needed and easy direct
monitoring of the reaction efficiency can be performed via Kaiser
test or test cleavage (see Note 11). To insert amide-to ester mutations, Boc-SPPS is the method of choice over Fmoc-SPPS, because
the basic N
α -deprotection step in Fmoc-SPPS can lead to hydrolysis
of the ester group.
Different resins are used for the synthesis of N- and C-terminal
fragments. C-terminal fragments do not need special requirements
for the resin and can be generated on commercially available resins
(see Note 12). For N-terminal fragments, the generation of a
C-terminal thioester is essential for the ligation reaction. Therefore,
functional resins are used, for example, comprising a hydrazine
linker in Fmoc-SPPS or thioester generating resins in Boc-SPPS.
3.7 Fmoc/t-Bu-SPPS
for the Synthesis
of Phosphopeptides
The protocol below, for Fmoc-SPPS is suitable for the synthesis of
peptides with canonical amino acids and for inserting modifications
like glycosylations and phosphorylations. We provide a standard
protocol for Fmoc-SPPS, including the example of the introduction of phospho-amino acid building blocks.
206
Christin Kossmann et al.
Cleavage to Generate
N-Terminal Cys
Protein Fragment
Δ C PDZ
1. Dialyze recombinant protein from pRSET(Δ C PDZ) against
1 L of factor Xa buffer at 4
C. Exchange dialysis buffer with
fresh buffer every 2 h for 3 times. Allow sample to dialyze
overnight after the last buffer exchange.
2. Transfer protein sample to a fresh 50 mL-conical tube. Add
factor Xa according to manufacturer’s instructions and allow
enzymatic cleavage to progress at 4
C on a rotating or rocking
table for 12–24 h. Remove 10 μL aliquots every hour and
monitor reaction with LC-MS (see Note 10).
3. Prepare a 5-mL His-trap FF column for purification by following Subheading 3.3.2, step 3 with cleavage buffer. Purify
cleavage mixture immediately after reaction is complete to
avoid nonspecific cleavage, using steps 4 and 5 in Subheading
3.4 with factor Xa cleavage buffer as the eluent.
4. Dialyze the final protein fragment against ultrapure water to
remove salt and buffer components, and lyophilize protein to
form white amorphous solids. Analyze compound with UPLC
and LC-MS, and store at À20
C until they are ready to be used
for processing.
3.6 Solid-Phase
Peptide Synthesis
SPPS can be performed with two protection techniques: Boc/Bzl
or Fmoc/t-Bu. The latter has the advantage of an orthogonal
protection system, which allows milder conditions and a broader
range in the pH-dependent reaction conditions for selective cleavage of side-chain protection groups and the Fmoc-group [3]. The
Fmoc-SPPS method is preferred over Boc-SPPS in regard to phosphorylated peptides because phosphate groups are not stable during HF cleavage. Several automated synthesizers are available,
enabling fast and convenient SPPS. For the inclusion of expensive
building blocks, such as phospho-amino acids, manual synthesis is
often preferred, because less material is needed and easy direct
monitoring of the reaction efficiency can be performed via Kaiser
test or test cleavage (see Note 11). To insert amide-to ester mutations, Boc-SPPS is the method of choice over Fmoc-SPPS, because
the basic N
α -deprotection step in Fmoc-SPPS can lead to hydrolysis
of the ester group.
Different resins are used for the synthesis of N- and C-terminal
fragments. C-terminal fragments do not need special requirements
for the resin and can be generated on commercially available resins
(see Note 12). For N-terminal fragments, the generation of a
C-terminal thioester is essential for the ligation reaction. Therefore,
functional resins are used, for example, comprising a hydrazine
linker in Fmoc-SPPS or thioester generating resins in Boc-SPPS.
3.7 Fmoc/t-Bu-SPPS
for the Synthesis
of Phosphopeptides
The protocol below, for Fmoc-SPPS is suitable for the synthesis of
peptides with canonical amino acids and for inserting modifications
like glycosylations and phosphorylations. We provide a standard
protocol for Fmoc-SPPS, including the example of the introduction of phospho-amino acid building blocks.
206
Christin Kossmann et al.