Peptide-Based Drug Design

18 Bulet
3. Centrifuge between 8000 and 12,000g (30 min, 4 ◦ C) to remove lipids (upper
phase), denaturized proteins, and debris. If necessary, the supernatant can be
clarified by an additional step with filter units (0.8-�m membranes, Millipore).
3.2.3. Extraction from Hemocytes, Epithelia, and Salivary Glands
1. Thaw the sample (hemocytes, epithelia) on ice if not freshly prepared.
2. The salivary glands are snap-frozen in liquid nitrogen.
3. Homogenize (see Note 7) using sonication (3 × 30 s) at medium power in the
presence of 2 M acetic acid (in an ice-cold water bath, a solution of 0.2 M acetic
acid is also an option to decrease the protein content of the extracted material).
Extract overnight at 4 ◦ C under gentle stirring in 2 M acetic acid supplemented with
a protease inhibitor (e.g., aprotinin).
4. Centrifuge between 8000 and 12,000g (30 min, 4 ◦ C) to separate the supernatant
(cytosolic fraction) from the organelle-rich fraction or tissue debris (pellet). If
necessary, the supernatant can be clarified by additional step with filter units
(0.8-�m membranes, Millipore).
3.3. Purification of the Natural Bioactive Peptides
Because of their well-recognized physicochemical properties (relatively
high hydrophobicity, cationic character, and short length) reversed-phase, sizeexclusion,
and cation exchange chromatographies are particularly appropriate to
purify bioactive peptides from the immune system of invertebrates. The sensitivity
of HPLC, MS, Edman degradation, and liquid growth inhibition assays
allow one to use from narrow (e.g., 2.1-mm internal diameter) down to microor
nano-columns.
3.3.1. Prepurification
To limit the co-precipitation of the peptides of interest with remaining
proteins, the first purification step is performed either by SPE on open-columns
or cartridges that supports large volumes of diluted extracts or using disposable
Ultrafree-CL (Millipore) ultrafiltration (UF) cartridge units (both strategies can
be combined, UF prior SPE or SPE prior UF; see ref. 28). Open columns can
be bought manufactured (e.g., Sep-Pak cartridges, WatersTM ) or handmade (bulk
phase available, e.g., from WatersTM ).
3.3.1.1. SOLID-PHASE EXTRACTION
This procedure has been already reported in ref. 22.Briefly:
1. Solvate the cartridge with methanol and equilibrate with acidified water (0.05%
TFA).
2. Load the acidified extract (pH < 4).