Peptide-Based Drug Design

Targeted Therapeutic and Imaging Agents 287
3. PBS pH 7.4: dilute 10X PBS concentrate (Gibco/BRL) with Milli-Q
deoxygenated water (see Note 1).
4. Tricine buffer (114 mM): dissolve 2 g N-[tris(hydroxymethyl)methyl]glycine
(Tricine, from Sigma) in 100 mL Milli-Q deoxygenated water (see Note 2).
5. SnCl2 solution: dissolve SnCl2·2H2O (Sigma) in 0.1 N HCl at 50 mg/mL.
6. Tin-Tricine reagent: dissolve 2.0 g Tricine in 97 mL of deoxygenated Milli-Q
water, adjust pH to 7.1 using approximately 1.3 mL of 1 N NaOH. Seal the flask
with a cannulated airtight septum and purge with nitrogen for 60 min. Add 1.2 mL
of SnCl2 solution, mix and transfer to nitrogen-filled septum-capped vials, 1 mL
in each, with a final composition of 20 mg tricine and 0.6 mg of SnCl2 at pH 7.1
(see Note 3).
7. Radionuclides: 99m TcO4 (5—10 mCi in a volume of approx 50 mL, GE
Healthcare, Sunnyvale, CA) and 64 Cu (5–10 mCi in 2–10 �L from Washington
University Medical School, St. Louis, MO).
8. PD-10 columns (GE Healthcare).
9. Indicator strips pH 4.0–7.0 (Merck, West Point, PA).
10. NaOOCCH3 buffer (1 M) pH 6.0: Dilute 3 M NaAc, pH 5.5 (Sigma) with Milli-Q
water and adjust pH to 6.0 with 0.1 N NaOH.
11. NaOOCCH3 buffer (0.1 M) pH 5.5: Dilute 3 M NaAc, pH 5.5 with Milli-Q water.
3. Methods
3.1. Recovery of scVEGF from Inclusion Bodies
1. To obtain 1 L of induced bacterial culture, grow E. coli BL21(DE3) bacteria
transfected with the pET/C4(G4S)/scVEGF plasmid in four 1-L flasks, each flask
containing 250 mL LB medium supplemented with 30 mg/mL of kanamycin.
Maintain the temperature at 37 ◦ C, shaking rate may vary from 220 to 300 rpm.
2. Once bacteria have reached an optical density of 0.4–0.6 optical units at 600
nm, induce scVEGF expression by adding 0.25 mL of 1 M IPTG to each flask.
Continue incubating at 37 ◦ C with 300 rpm shaking for 2 h.
3. Harvest 1 L of induced bacterial culture by centrifugation at 4400 g for 30 min
at 4 ◦ C (Beckman J2-21 centrifuge, Beckman Coulter, Fullerton, CA). Carefully
remove and discard the supernatant. Resuspend bacterial pellet in 1X PBS using
a 10-mL disposable plastic pipet. Pipet the solution up and down until no visible
pellet is left in the suspension. Adjust the final volume to 25 mL with 1X PBS.
4. Pass bacterial suspension twice through high-pressure homogenizer (EmulsiFlex-
C5, Avestin, Canada) at 10,000–15,000 psi.
5. Split homogenized bacteria in two 50-mL sterile centrifuge tubes and centrifuge
at 12,000 g for 15 min at 4 ◦ C to separate the soluble part of bacterial lysate and
inclusion body fraction. Discard the supernatants, trying not to disturb the pellet
(inclusion bodies).
6. Resuspend each pellet in 2 mL of ice-cold high-salt wash buffer by pipetting up
and down several times. Once the pellet is resuspended, adjust solution volume
to 30 mL in each tube, using the same ice-cold buffer.