The authors would like to thank Eleanor Fleming and Jessica Chen for helpful discussions. Funding was provided by National Institute of Allergy and Infectious Diseases (NIH-NIAID) Award R15AI079685 and a Research Corporation Cottrell College Science Award. Additional student support was provided by the Howard Hughes Medical Institute and the Staley fund.

1. Preparation of Large Unilamellar Lipid Vesicles (LUVs)
<ol>
 <li>Prepare LUVs to serve as cell membrane mimics for the assay19.</li>
 <li>Mix phosphatidylcholine (POPC, 760.10 g/mol), phosphatidylglycerol (POPG. 770.99 g/mol), 5-dimethylaminonaphthalene-1-sulfonyl phosphatidylethanolamine (DNS-POPE, 994.350 g/mol) (Avanti Polar Lipids) dissolved in chloroform in a 50:45:5 ratio. As every assay requires the separate preparation of both experimental and control LUVs, two lipid cake vials should be prepared....

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No conflicts of interest declared.

The protocol presented here can be used to assess the relative change in the concentration of peptides inside and outside of lipid vesicles. These changes are related to translocation ability. This protocol can be used to identify cell-penetrating peptides with potential as drug delivery vectors. As interest in cell-penetrating peptides grows, it will be interesting to see how methods that directly measure the translocation event are developed and used in a quantitative manner.
In our lab, we...

<table border="1">
<tbody>
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
</tr>
<tr>
<td>16:0-18:1 PG</td>
<td>Avanti Polar Lipids</td>
<td>840457C</td>
</tr>
<tr>
<td>1:18 Dansyl PE</td>
<td>Avanti Polar Lipids</td>
<td>810330C</td>
</tr>
<tr>
<td>16:0-18:1 PC</td>
<td>Avanti Polar Lipids</td>
<td>850457C </td>
</tr>
<tr>
<td>Porcine trypsin</td>
<td>Sigma</td>
<td>T-0303</td>
</tr>
<tr>
<td>Bowman-Birk trypsin/chymotrypsin inhibitor</td>
<td>Sigma</td>
<td>T-9777</td>
</tr>
<tr>
<td>Mini-extruder</td>
<td>Avanti Polar Lipids</td>
<td>610000</td>
</tr>
<tr>
<td>Ammonium molybdate (para)</td>
<td>Alfa Aesar</td>
<td>10811</td>
</tr>
<tr>
<td>L-ascorbic acid</td>
<td>Sigma</td>
<td>A1417</td>
</tr>
<tr>
<td>Hydrogen Peroxide, 30% solution</td>
<td>Mallinckrodt chemicals</td>
<td>5240-05</td>
</tr>
<tr>
<td>HEPES</td>
<td>Sigma</td>
<td>H-3375</td>
</tr>
<tr>
<td>NaCl</td>
<td>Sigma</td>
<td>S-9625</td>
</tr>
<tr>
<td>EDTA</td>
<td>Sigma</td>
<td>E5134</td>
</tr>
<tr>
<td>NaH2PO4</td>
<td>Sigma</td>
<td>S-0751</td>
</tr>
</tbody>
</table>

measuring peptide translocation into large unilamellar vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

Watch this Scientific Journal Video about Measuring Peptide Translocation into Large Unilamellar Vesicles at JoVE.com

Measuring Peptide Translocation into Large Unilamellar Vesicles

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

measuring-peptide-translocation-into-large-unilamellar-vesicles

Research

JoVE Journal

Biology

13.7K visualizações.  Wellesley College. Este protocolo de detalhes um método para a medida quantitativa da translocação de peptídeos em grandes vesículas lipídicas unilamelares. Este método também fornece informações sobre a taxa de translocação de membrana e pode ser usado para identificar peptídeos que de forma eficiente e espontânea cruz bicamadas lipídicas.