Antimicrobial peptides

 Antimicrobial Peptides :

1. Introduction to AMPs :

★AMPs are small, naturally occurring proteins (typically 10–100 amino acids) that play a crucial role in innate immunity.

★They exhibit broad-spectrum activity against bacteria, fungi, viruses, and even cancer cells.

★Common examples: Defensins, Cathelicidins, Magainins, LL-37.

2. Sources of AMPs :

Natural Sources:

Animals: Skin, epithelial cells, neutrophils (e.g., defensins from humans).

Plants: Thionins, defensins.

Microorganisms: Bacteriocins from bacteria.

Marine organisms: Tunicates, sponges, and mollusks.


Synthetic Sources:

Designed based on known AMP structures or generated through bioinformatics tools.

3. Methods of Production

A. Chemical Synthesis

★Solid Phase Peptide Synthesis (SPPS):

★Uses stepwise addition of amino acids on a resin.

★Suitable for peptides <50 amino acids.

★High purity but expensive and less efficient for long peptides.


Advantages:

>High purity.

>Incorporation of modified amino acids.


Disadvantages:

>High cost.

>Not ideal for large-scale production.

B. Recombinant Expression Systems

Used to produce larger quantities at lower cost.

1. Bacterial Systems (e.g., E. coli)

>Most common system.

>AMPs often toxic to host, so fusion proteins are used.

>Requires cleavage and purification.



2. Yeast Systems (e.g., Pichia pastoris)

>Secretion of peptides into medium.

>Better folding and post-translational modifications.

3. Insect Cells (Baculovirus System)

>High expression levels.

>Suitable for peptides needing complex modifications.

4. Mammalian Cells

>Expensive, used when correct folding and PTMs are essential.

5. Plant-based Systems

>Cost-effective and safe.

>Transgenic plants can produce AMPs in seeds or leaves.


4. Fusion Tags for AMP Production

>Fusion tags improve expression, solubility, and protect the host from AMP toxicity.

>Common tags: GST, MBP, His-tag, SUMO.

>Cleaved post-expression using proteases (e.g., thrombin, TEV).

5. Purification of AMPs

>Affinity Chromatography: Using tags like His-tag (Ni-NTA column).

>Ion Exchange Chromatography: Based on charge differences.

>Reverse-Phase HPLC: For final purification and analysis.

>Size-Exclusion Chromatography: For size-based separation.

6. Characterization of AMPs

>Mass Spectrometry (MS) – Molecular weight.

>Circular Dichroism (CD) Spectroscopy – Secondary structure.

>NMR / X-ray Crystallography – 3D structure.

>Antimicrobial Assays – MIC determination against microbes.

7. Challenges in AMP Production

>Cytotoxicity to host cells.

>Proteolytic degradation.

>Low expression yields.

>High cost of synthesis/purification.


8. Future Prospects and Trends

>In silico design of synthetic AMPs with enhanced stability and activity.

>Peptidomimetics: Synthetic analogs with improved properties.

>Encapsulation and delivery strategies to improve AMP stability in vivo.


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