बड़े Unilamellar vesicles में पेप्टाइड स्थानान्तरण माप
Summary
यह प्रोटोकॉल बड़े unilamellar लिपिड vesicles में पेप्टाइड स्थानान्तरण के मात्रात्मक उपाय के लिए एक विधि के विवरण. इस विधि भी झिल्ली स्थानान्तरण की दर के बारे में जानकारी प्रदान करता है और पेप्टाइड्स है कि कुशलतापूर्वक और अनायास लिपिड bilayers पार की पहचान करने के लिए इस्तेमाल किया जा सकता है.
Abstract
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.
Protocol
Discussion
यहाँ प्रस्तुत प्रोटोकॉल लिपिड vesicles के अंदर और बाहर पेप्टाइड्स की एकाग्रता में रिश्तेदार परिवर्तन का आकलन करने के लिए इस्तेमाल किया जा सकता है. इन परिवर्तनों को स्थानान्तरण करने की क्षमता से संबंधित ह?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों उपयोगी विचार – विमर्श के लिए Eleanor फ्लेमिंग और जेसिका चेन धन्यवाद देना चाहूंगा. अनुदान एलर्जी और संक्रामक रोगों के राष्ट्रीय संस्थान (एनआईएच NIAID) के द्वारा R15AI079685 पुरस्कार और अनुसंधान निगम Cottrell कॉलेज विज्ञान पुरस्कार प्रदान किया गया. अतिरिक्त छात्र समर्थन हॉवर्ड ह्यूजेस मेडिकल इंस्टीट्यूट और Staley फंड द्वारा प्रदान की गई थी.
Materials
| Name of the reagent | Company | Catalogue number |
| 16:0-18:1 PG | Avanti Polar Lipids | 840457C |
| 1:18 Dansyl PE | Avanti Polar Lipids | 810330C |
| 16:0-18:1 PC | Avanti Polar Lipids | 850457C |
| Porcine trypsin | Sigma | T-0303 |
| Bowman-Birk trypsin/chymotrypsin inhibitor | Sigma | T-9777 |
| Mini-extruder | Avanti Polar Lipids | 610000 |
| Ammonium molybdate (para) | Alfa Aesar | 10811 |
| L-ascorbic acid | Sigma | A1417 |
| Hydrogen Peroxide, 30% solution | Mallinckrodt chemicals | 5240-05 |
| HEPES | Sigma | H-3375 |
| NaCl | Sigma | S-9625 |
| EDTA | Sigma | E5134 |
| NaH2PO4 | Sigma | S-0751 |
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