3. Sterilized Luria Broth (pH 7.2): dissolve premixed LB broth in
1 L of ultrapure water according to manufacturer’s instructions
and autoclave at 121
C for 30 min.
4. Sterilized 300 mL and 5 L culture flasks, autoclaved at 121
C
for 30 min.
5. 50 mg/mL ampicillin stock solution in ultrapure water.
6. 1 M IPTG stock solution in ultrapure water.
2.3 Protein
Purification
1. Elution buffer (50 mL): 50 mM sodium phosphate pH 7.4,
250 mM imidazole.
2. HisTrap Fast Flow (FF) columns, 5 mL.
3. Lysis Buffer (50 mL): 50 mM sodium phosphate pH 7.4,
10 mM MgCl 2 , 25 μg/mL DNase, complete protease inhibitor
(1 tablet/50 mL). Add DNase and protease inhibitor right
before use.
4. Wash buffer (200 mL): 50 mM sodium phosphate pH 7.4,
25 mM imidazole.
2.4 Thioester
Generation
1. Thiolysis buffer: 1–2 M urea, 50 mM sodium phosphate
pH 6.8, 150 mM NaCl.
2. Dialysis membrane or device, with molecular weight cutoff
(MWCO) of 10 kDa.
3. Sodium 2-mercaptoethanesulfonate (MESNa).
2.5 Factor Xa
Cleavage
1. Cleavage buffer: 50 mM Tris–HCl pH 8, 100 mM NaCl, 6 mM
calcium chloride.
2. Factor Xa protease.
3. Dialysis membrane or device, with a MWCO of 10 kDa.
Table 1
Primers used for the construction of plasmids encoding recombinant EPL fragments
Forward
Reverse
1. Primers for introduction of factor Xa site
placed adjacent to the N-terminal Cys
a,b
5
0 -(NNN) 5 ATC GAG
GGA AGG TGC
(NNN) 5 -3´
5
0 -(NNN) 5 GCA CCT
TCC CTC GAT
(NNN) 5 -3´
2. Primers for PDZ cloning into pTWIN1
c,d 5
0 -GGA ATT CAT ATG
(NNN) 10 -3´
5
0 -GGT GGT TGC TCT
TCC (NNN) 6 -3´
a
Bold letters highlight the gene encoding factor Xa site and a Cys residue
b
(NNN) x corresponds to flanking codons from the target gene, with x being the number of codons
c
NdeI site is underlined in the sequence
d
SapI site is italicized in the sequence
Synthetic PDZ Domains
199