Name: Productie en testen van antimicrobiële peptiden en hun nabootsers (Video) | JoVE Journal
Uploaded: 2026-04-10T13:08:00.000+00:00
Duration: 10 min 35 s
Description: Leer de productie en evaluatie van antimicrobiële peptiden en hun synthetische nabootsers. Dit protocol integreert synthese, MIC/MBC-bepalingen en cytotoxiciteitstests met Mel4 en RK-758 voor de ontwikkeling van nieuwe anti-infectieve middelen.

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<li>Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptides+chemistry%2C+manufacturing%2C+and+controls%5BTitle%5D)+AND+%22Peptide+Science%3A+Chemical+Ligation%2C+Lead+Generation%2C+and+Therapeutic+Advances%22%5BJournal%5D)">Peptides chemistry, manufacturing, and controls.</a> Peptide Science: Chemical Ligation, Lead Generation, and Therapeutic Advances. , 471-508 (2025).</li>
<li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+and+purification+of+antimicrobial+peptide+buforin+IIb+in+Escherichia+coli%5BTitle%5D)+AND+%22Biotechnol.+Lett%22%5BJournal%5D)">Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli.</a> Biotechnol. Lett. 33 (11), 2121-2126 (2011).</li>
<li>Cai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide+synthesis%3A+a+review+of+classical+and+emerging+methods%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">Peptide synthesis: a review of classical and emerging methods.</a> Biofabrication. 18 (1), 012001(2026).</li>
<li>Gentilucci, L., De Marco, R., Cerisoli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+modifications+designed+to+improve+peptide+stability%3A+incorporation+of+non-natural+amino+acids%2C+pseudo-peptide+bonds%2C+and+cyclization.%C2%A0Curr.+Pharm+pseudo-peptide%5BTitle%5D)+AND+%22Des%22%5BJournal%5D)">Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Curr. Pharm pseudo-peptide.</a> Des. 16 (28), 3185-3203 (2010).</li>
<li>Kuppusamy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tuning+the+anthranilamide+peptidomimetic+design+to+selectively+target+planktonic+bacteria+and+biofilm%5BTitle%5D)+AND+%22Antibiotics%22%5BJournal%5D)">Tuning the anthranilamide peptidomimetic design to selectively target planktonic bacteria and biofilm.</a> Antibiotics. 12 (3), 585(2023).</li>
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<li>Wiegand, I., Hilpert, K., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agar+and+broth+dilution+methods+to+determine+the+minimal+inhibitory+concentration+%28MIC%29+of+antimicrobial+substances%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.</a> Nat. Protoc. 3 (2), 163-175 (2008).</li>
<li>Ji, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adsorption+and+morphology+analysis+of+bovine+serum+albumin+on+a+micropillar-enhanced+quartz+crystal+microbalance%5BTitle%5D)+AND+%22J.+Phys.+Chem.+B%22%5BJournal%5D)">Adsorption and morphology analysis of bovine serum albumin on a micropillar-enhanced quartz crystal microbalance.</a> J. Phys. Chem. B. 128 (41), 10247-10257 (2024).</li>
<li>Yasir, M., Dutta, D., Willcox, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+antimicrobial+peptides+and+ciprofloxacin+against+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Activity of antimicrobial peptides and ciprofloxacin against Pseudomonas aeruginosa biofilms.</a> Molecules. 25 (17), (2020).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+determination+of+susceptibility+of+bacteria+to+antimicrobial+agents.+terminology%5BTitle%5D)+AND+%22Clin.+Microbiol.+Infect%22%5BJournal%5D)">Methods for the determination of susceptibility of bacteria to antimicrobial agents. terminology.</a> Clin. Microbiol. Infect. 4 (5), 291-296 (1998).</li>
<li>Saetan, T., Sukwattanasinitt, M., Wacharasindhu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mild+photocatalytic+synthesis+of+guanidine+from+thiourea+under+visible+light%5BTitle%5D)+AND+%22Org.+Lett%22%5BJournal%5D)">A mild photocatalytic synthesis of guanidine from thiourea under visible light.</a> Org. Lett. 22 (20), 7864-7869 (2020).</li>
<li>Woodring, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disrupting+the+conserved+salt+bridge+in+the+trimerization+of+influenza+A+nucleoprotein%5BTitle%5D)+AND+%22J.+Med.+Chem%22%5BJournal%5D)">Disrupting the conserved salt bridge in the trimerization of influenza A nucleoprotein.</a> J. Med. Chem. 63 (1), 205-215 (2019).</li>
<li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+bacterial+expression+systems+for+heterologous+protein+production%3A+from+molecular+and+biochemical+fundamentals+to+commercial+systems%5BTitle%5D)+AND+%22Appl.+Microbiol.+Biotechnol%22%5BJournal%5D)">Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.</a> Appl. Microbiol. Biotechnol. 72 (2), 211-222 (2006).</li>
<li>Berridge, M. V., Herst, P. M., Tan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tetrazolium+dyes+as+tools+in+cell+biology%3A+new+insights+into+their+cellular+reduction%5BTitle%5D)+AND+%22Biotechnol.+Annu.+Rev%22%5BJournal%5D)">Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.</a> Biotechnol. Annu. Rev. 11, 127-152 (2005).</li>
<li>Deni, W. H., Gao, T., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+reconstituting+peptides%2Fpeptidomimetics+from+DMSO+to+aqueous+buffers+for+circular+dichroism+analyses%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for circular dichroism analyses.</a> STAR Protoc. 5 (1), 102850(2024).</li>
<li>Lou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+production+systems+and+optimization+strategies+of+antimicrobial+peptides%3A+a+review%5BTitle%5D)+AND+%22World+J.+Microbiol.+Biotechnol%22%5BJournal%5D)">Microbial production systems and optimization strategies of antimicrobial peptides: a review.</a> World J. Microbiol. Biotechnol. 41 (2), 66(2025).</li>
<li>Hancock, R. E., Sahl, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+and+host-defense+peptides+as+new+anti-infective+therapeutic+strategies%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.</a> Nat. Biotechnol. 24 (12), 1551-1557 (2006).</li>
</ol>

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<table><tbody><tr><td>1,2-ethanedithiol</td><td>Oakwood </td><td>M03555</td><td></td></tr><tr><td>100X Antibiotics</td><td>Sigma Aldrich</td><td>A5955-20ML</td><td></td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride</td><td>Combi Blocks</td><td>SS-7536</td><td>Verder gerefereerd als EDC</td></tr><tr><td>2-Naphthoyl chloride</td><td>Combi Blocks</td><td>OR-0173</td><td></td></tr><tr><td>Acetic acid </td><td>Ajax Finechem</td><td>R18401O</td><td></td></tr><tr><td>Acetonitrile</td><td>VWR </td><td>20060.35</td><td></td></tr><tr><td>Alkaline phosphatase</td><td>New England Biolabs</td><td>M0525S</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A1933</td><td></td></tr><tr><td>Cation-adjusted Mueller-Hinton Broth (CAMHB)</td><td>Becton, Dickinson and Company</td><td>212322</td><td></td></tr><tr><td>Cbz-L-Trp</td><td>Combi Blocks</td><td>QB-6691</td><td></td></tr><tr><td>Colistin </td><td>Sigma Aldrich</td><td>C4461</td><td></td></tr><tr><td>Dey-Engley (D/E) Neutralising Broth</td><td>Oxoid</td><td>R453042</td><td></td></tr><tr><td>Diatomaceous earth (Celite 545)</td><td>Sigma Aldrich</td><td>102693</td><td>0.02-0.1 mm deeltjesgrootte</td></tr><tr><td>Dichloromethane (DCM)</td><td>VWR </td><td>23366.327</td><td></td></tr><tr><td>Diethyl ether</td><td>Chem-Supply</td><td>EA036</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Chem-Supply Pty Ltd </td><td>LCS-CHEM-0098</td><td></td></tr><tr><td>Dithiothreitol(DTT)</td><td>Thermo Scientific</td><td>J15397.03</td><td></td></tr><tr><td>DMEM culture medium</td><td>Thermo Fisher Scientific </td><td>UCS-BIO-0058</td><td></td></tr><tr><td>E. coli BL21(DE3) host</td><td>Sigma Aldrich</td><td>70235-M</td><td></td></tr><tr><td>Electroporator</td><td>Thermo Scientific</td><td>NEON1SK</td><td></td></tr><tr><td>Ethyl acetate</td><td>Chem-Supply</td><td>EA011-2.5L-P</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Thermo Scientific</td><td>118432500</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>12003C-500ML</td><td></td></tr><tr><td>His-tagged TEV Protease </td><td>Sigma Aldrich</td><td>T4455</td><td></td></tr><tr><td>Hydroxybenzotriazole Hydrate</td><td>Chem-Implex</td><td>24755</td><td></td></tr><tr><td>Imidazole</td><td>Thermo Scientific</td><td>288-32-4</td><td></td></tr><tr><td>Isopropyl β-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma Aldrich</td><td>I6758</td><td></td></tr><tr><td>Luria-Bertani Broth (LB), </td><td>Thermo Scientific</td><td>12795027</td><td></td></tr><tr><td>Lysis Buffer </td><td>Thermo Scientific</td><td>89822</td><td></td></tr><tr><td>Mercury (II) Chloride</td><td>Sigma Aldrich</td><td>215465</td><td></td></tr><tr><td>Methanol (HPLC Grage)</td><td>VWR</td><td>A452-4K</td><td></td></tr><tr><td>MTT reagent</td><td>Sigma Aldrich</td><td>M2128-100MG</td><td></td></tr><tr><td>N,N-Diisopropylethylamine (DIPEA)</td><td>Sigma Aldrich</td><td>3440</td><td></td></tr><tr><td>N,N-Dimethyl formamide (DMF)</td><td>Chem-Supply</td><td>RP1051</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>ND-ONE</td><td></td></tr><tr><td>N-Boc-ethylenediamine</td><td>Combi blocks</td><td>AM-1613</td><td></td></tr><tr><td>NTA Affinity Resin Column</td><td>Sigma Aldrich</td><td>70666</td><td></td></tr><tr><td>Palladium on carbon</td><td>Sigma Aldrich</td><td>205699</td><td>10% w/w Pd</td></tr><tr><td>Plasmid Purification kit</td><td>QIAGEN</td><td>27104</td><td></td></tr><tr><td>Polypropylene microtiter tubes </td><td>SARSTEDT </td><td>72.690.008</td><td></td></tr><tr><td>Silica Gel for flash column chromatography</td><td>Silicycle</td><td>R12030B</td><td>Silica Gel 60, 230-400 mesh </td></tr><tr><td>Silica gel Thin layer chromatography plate</td><td>Merk</td><td>1055540001</td><td>0.2 mm laagdikte op aluminium achterkant met 254 nm fluorescente indicator</td></tr><tr><td>Sodium phosphate, 0.2M buffer soln</td><td>Thermo Scientific</td><td>J63816.AP</td><td></td></tr><tr><td>Sterile flat-bottomed 96-well polypropylene microtiter plate with lid</td><td>Corning</td><td>3599</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>Triethylamine (Et3N)</td><td>Chem-Supply</td><td>TR02151000</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Sigma Aldrich</td><td>T6508</td><td></td></tr><tr><td>Tris-HCL</td><td>Thermo Scientific</td><td>J62848.AK</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific</td><td>A16046.AE</td><td></td></tr><tr><td>Trypan blue</td><td>MP BIOMEDICALS AUSTRALIA PTY LTD</td><td></td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Sigma Aldrich</td><td>T4049-500ML</td><td></td></tr><tr><td>Trypticase soya agar </td><td>Becton, Dickinson and Company</td><td>211043</td><td></td></tr><tr><td>Urea </td><td>Thermo Scientific</td><td>29700</td><td></td></tr><tr><td>Vector (T7 promoter)</td><td>-</td><td></td><td>high-copy number plasmid naar keuze</td></tr></tbody></table>
```

productie en testen van antimicrobiële peptiden en hun nabootsers

Production and Testing of Antimicrobial Peptides and Their Mimics

Productie en testen van antimicrobiële peptiden en hun nabootsers

Antimicrobial peptides (AMPs) and their synthetic mimics are emerging as promising alternatives to conventional antibiotics, particularly in response to ...

production-and-testing-of-antimicrobial-peptides-and-their-mimics

Research

JoVE Journal

Biology

Biologie

Peptide tertiaire amides (PTA) zijn een superfamilie van peptidomimetica die omvatten maar zijn niet beperkt tot, peptiden, peptoïden en N-gemethyleerd peptiden. Hier beschrijven we een synthetische methode die zowel split-en-zwembad en sub-monomeer strategieën combineert tot een kraal een-verbinding bibliotheek van PTA synthetiseren.

Split-en-zwembad Synthese en karakterisatie van peptide Tertiaire amide Bibliotheek

Een eenvoudige en algemene methode voor de synthese van cyclische peptiden met behulp van microgolfbestraling wordt geschetst. Deze procedure maakt de synthese mogelijk van cyclische peptiden met een ruggengraat met verschillende conformatieën, terwijl de zijketens en de farmacofore groepen behouden blijven en daarom, maakt het mogelijk om te screenen op de bioactieve conformatie.

Ontwikkeling van een Backbone cyclisch peptide bibliotheek als potentieel antiparasitaire Therapeutics behulp van microgolfstraling

Hier presenteren we protocollen om snel enzymen te screenen en te karakteriseren op antimicrobiële activiteit. De microslide-diffusietest en de kleurstof-releasetest maken gebruik van doelbacteriële substraten voor kwalitatieve en kwantitatieve evaluatie van enzymatische activiteit.

Kwalitatieve en Kwantitatieve Testen voor detectie en karakterisering van proteïne Antimicrobials

Een protocol voor het synthetiseren van peptoïden met gemengde kationische functionaliteit in dezelfde sequentie wordt gepresenteerd (lysine- en arginine-achtige monomeren). Het daaropvolgende testen van deze verbindingen tegen Leishmania mexicana, de protozoa-parasieten die cutane leishmaniasis veroorzaken, wordt ook beschreven.

Een efficiënte methode voor de synthese van peptoïden met Gemengde lysine-type / Arginine-type monomeren en evaluatie van hun anti-leishmania Activity

Een eenvoudig en flexibel Bouillon microdilution methode voor het screenen van antischimmel verbindingen en extracten.

Bouillon Microdilution In Vitro Screening: een gemakkelijke en snelle methode om te ontdekken van nieuwe antischimmel Compounds

Het protocol stelt de Escherichia coli-selectieve druk opneming van niet-canonieke aminozuren (ncAAs) in het lactococcal antimicrobiële peptide nisine gebaseerd. De eigenschappen kunnen worden gewijzigd tijdens de recombinante expressie via substitutie met de gewenste ncAAs in de gedefinieerde groei media. Resulterende mutaties in de topicale worden toegewezen door groei remming testen en fluorescentie microscopie.

Antimicrobiële peptiden geproduceerd door selectieve druk opneming van niet-canonieke aminozuren

Hier presenteren we een protocol voor de productie van gramnegatieve Escherichia coli (E. coli) spheroplasts en gram-positieve Bacillus megaterium (B. megaterium) protoplasten duidelijk visualiseren en snel karakteriseren Peptide-bacteriën interacties. Dit biedt een systematische methode om te definiëren membraan lokaliseren en translocating peptiden.

Productie en visualisatie van bacteriële Spheroplasts en protoplasten te karakteriseren antimicrobiële Peptide lokalisatie

Het recombinante antilichaameiwit dat tot expressie komt in pIRES2-ZSGreen1-rAbs-APN-CHO-cellen en monoklonale antilichamen geproduceerd met behulp van traditionele hybridoma-technologie kan het varkensaminopeptidase N (APN) -eiwit herkennen en eraan binden.

Productie van monoklonale antilichamen gericht tegen aminopeptidase N in het varkensdarmslijmvliesepitheel

Dit protocol beschrijft een methode voor het verkrijgen van kwantitatieve gegevens over de antischimmelactiviteit van peptiden en andere verbindingen, zoals antischimmelmiddelen met kleine moleculen, tegen Candida albicans. Het gebruik van optische dichtheid in plaats van het tellen van kolonievormende eenheden om groeiremming te kwantificeren, bespaart tijd en middelen.

Kwantificeren van de antischimmelactiviteit van peptiden tegen Candida albicans

We presenteren de procedures aan te tonen dat liganden binden aan het oppervlak membraan van de protozoa cellulose verterende protozoa in de darm van de Formosan ondergrondse termieten met behulp van fluorescentie microscopie en dat liganden gekoppeld aan lytische peptiden kill...

Testen Protozoacidal Activiteit van Ligand-lytische Peptiden Against Protozoa Termite Gut In vitro (Protozoa Cultuur) en In vivo (Micro-injectie in Termite einddarm)

Leer de productie en evaluatie van antimicrobiële peptiden en hun synthetische nabootsers. Dit protocol integreert synthese, MIC/MBC-bepalingen en cytotoxiciteitstests met Mel4 en RK-758 voor de ontwikkeling van nieuwe anti-infectieve middelen.

Productie en testen van antimicrobiële peptiden en hun nabootsers

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