Peptide-Based Drug Design

16 Bulet
7. Small-volume low-protein absorption polypropylene tubes—either Eppendorf or
Minisorp NUNC Immuno tubes.
8. Water bath or dry incubator adjusted at 45 ◦ C.
2.6. Antimicrobial Assays
Bacteria, yeast, and fungi should be purchased from certified institutions such
as ATCC or Pasteur Institute or from private collections. For antimicrobial activity
screening during the course of purification, sensitive strains are recommended:
Micrococcus luteus (Gram-positive), Escherichia coli D31 (Gram-negative, see
Note 2), and Neurospora crassa (filamentous fungus) are good test organisms.
Pathogenic strains for the animal model can also be used (29). The liquid growth
inhibition assays (antibacterial and antifungal) are preferred to the radial diffusion
assays for high-throughput screening (HTS). In addition, the liquid growth
inhibition assays allow evaluating within a same experiment growth inhibition and
colony-forming unit (CFU) counting, a method frequently used for discriminating
between bacteriostatic and bacteriolytic activities.
1. Adapted media for the microbial strains: potato dextrose broth (PDB, Difco) used
at half-strength (12 g/L for filamentous fungi supplemented with antibiotics) and
Poor broth (PB, 1% bactotryptone, 0.9% NaCl w/v, pH 7.4) if bacterial strains for
activity screening during isolation.
2. Luria Bertani broth (LB, 1% bactotryptone, 0.5% yeast extract, 0.9% NaCl w/v,
pH 7.4) for CFU counting.
3. Antibiotics: 10 mg/mL tetracyclin (Sigma) in dimethylsulfoxide (DMSO) and 100
�g/mL cefotaxim (Sigma) in water.
4. 96-well microtitration plates for cell culture (Nunc) and microplate reader and a
spectrophotometer equipped with a filter between 580 and 620 nm.
3. Methods
The following protocols can be used for the isolation and structural characterization
of any natural bioactive peptides from the immune system of invertebrates.
The different procedures that will be detailed below refer to the
identification and primary structure determination of the Drosophila immuneinduced
peptides (19,20,23,27,30) and of bioactive peptides from the immune
system of other Diptera (17,21,24,31). These approaches were also successfully
used for the discovery of bioactive peptides from crustaceans, arachnids,
and mollusks. These methods should be considered as a guideline and not as
the exact procedure to follow (see Note 3). The suggested procedures will
be reported following the normal order of execution, (1) induction of the
immune response by an experimental infection, (2) collection of the immunocompetent
cells (hemocytes), tissues (epithelia, trachea, salivary glands, etc.)