Fmoc-SPPS via the hydrazine method are described along with the
expression of C-terminal thioesters via intein fusion proteins and
N-terminal Cys containing proteins. PDZ domains inconveniently
lack a Cys residue where we desire to place a ligation junction, but
we circumvent this by employing radical desulfurization, which will
also be described in these protocols.
2 Materials
2.1 Plasmid
Construction
1. Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible bacterial expression vector with ampicillin resistance that encodes
the codon-optimized DNA construct of the target protein
fused to a polyhistidine purification tag, for example pRSET
(PDZ) (see Note 1).
2. Intein-encoding plasmid with ampicillin resistance for IPTG
inducible bacterial expression, for example pTWIN1.
3. 10 μM oligonucleotide primer stock solutions in nuclease-free
water (see Table 1).
4. Site-directed mutagenesis kit based on a high-fidelity DNA
polymerase.
5. Nuclease-free water.
6. Chemically competent E. coli cloning strain (TOP10).
7. S.O.C medium.
8. DNA miniprep kit.
9. 50 mg/mL ampicillin stock solution in ultrapure water.
10. Sterile Luria Broth (LB) agar plates: mix LB agar into ultrapure
water. Autoclave at 121
C for 30 min and cool down to 50
C
before adding ampicillin (100 μg/mL) and distributing on
92 mm x 16 mm sterile clear petri dishes. Store plates at 4
C.
11. Sterile Luria Broth (LB) media.
12. High-fidelity DNA polymerase and appropriate buffers.
13. NdeI and SapI restriction enzymes.
14. TAE buffer: 1 M ethylenediamine tetraacetic acid, 0.04 M Trisbase, 0.01 M acetic acid.
15. 1.5% agarose gel, prepared by melting solid agarose (ultrapure
agarose) suspended in TAE buffer using a microwave oven.
16. DNA gel purification kit.
17. T4 DNA ligase and appropriate buffers.
2.2 Protein
Expression
1. Chemically competent E. coli expression strain (BL21
dE3 pLys).
2. Sterile LB agar plates (see Subheading 2.1).
198
Christin Kossmann et al.
expression of C-terminal thioesters via intein fusion proteins and
N-terminal Cys containing proteins. PDZ domains inconveniently
lack a Cys residue where we desire to place a ligation junction, but
we circumvent this by employing radical desulfurization, which will
also be described in these protocols.
2 Materials
2.1 Plasmid
Construction
1. Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible bacterial expression vector with ampicillin resistance that encodes
the codon-optimized DNA construct of the target protein
fused to a polyhistidine purification tag, for example pRSET
(PDZ) (see Note 1).
2. Intein-encoding plasmid with ampicillin resistance for IPTG
inducible bacterial expression, for example pTWIN1.
3. 10 μM oligonucleotide primer stock solutions in nuclease-free
water (see Table 1).
4. Site-directed mutagenesis kit based on a high-fidelity DNA
polymerase.
5. Nuclease-free water.
6. Chemically competent E. coli cloning strain (TOP10).
7. S.O.C medium.
8. DNA miniprep kit.
9. 50 mg/mL ampicillin stock solution in ultrapure water.
10. Sterile Luria Broth (LB) agar plates: mix LB agar into ultrapure
water. Autoclave at 121
C for 30 min and cool down to 50
C
before adding ampicillin (100 μg/mL) and distributing on
92 mm x 16 mm sterile clear petri dishes. Store plates at 4
C.
11. Sterile Luria Broth (LB) media.
12. High-fidelity DNA polymerase and appropriate buffers.
13. NdeI and SapI restriction enzymes.
14. TAE buffer: 1 M ethylenediamine tetraacetic acid, 0.04 M Trisbase, 0.01 M acetic acid.
15. 1.5% agarose gel, prepared by melting solid agarose (ultrapure
agarose) suspended in TAE buffer using a microwave oven.
16. DNA gel purification kit.
17. T4 DNA ligase and appropriate buffers.
2.2 Protein
Expression
1. Chemically competent E. coli expression strain (BL21
dE3 pLys).
2. Sterile LB agar plates (see Subheading 2.1).
198
Christin Kossmann et al.