Name: extraction and visualization of protein aggregates after treatment of escherichia coli with a proteotoxic stressor
Uploaded: 2021-06-29T13:30:00
Description: Watch this Scientific Journal Video about Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor at JoVE.com

This work was supported by Illinois State University School of Biological Sciences startup funds, Illinois State University New Faculty Initiative Grant, and the NIAID grant R15AI164585 (to J.-U. D.). G.M.A. was supported by the Illinois State University Undergraduate Research Support Program (to G.M.A.). K. P. H. was supported by a RISE fellowship provided by the German Academic Exchange Service (DAAD). The authors thank Dr. Uwe Landau and Dr. Carsten Meyer from Largentech Vertriebs GmbH for providing the AGXX powder. Figures 1, Figure 2, Figure 3, Figure 4, and Figure 5 were generated with Biorender.

1. Stress treatment of E. coli strains MG1655 and CFT073
<ol>
	<li>Inoculate 5 mL of lysogeny broth (LB) medium with a single colony of commensal E. coli strain MG1655 and uropathogenic E. coli (UPEC) strain CFT073, respectively, and incubate for 14-16 h (overnight) at 37 &#176;C and 300 rpm. 
	NOTE: Escherichia coli CFT073 is a human pathogen. Handling of CFT073 must be performed with appropriate biosafety measures in a Biosafety Level-2 certified lab.</li>
	<li>Dilute each str...

<ol>
	<li>Dahl, J. -. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HdeB+functions+as+an+acid-protective+chaperone+in+bacteria.">HdeB functions as an acid-protective chaperone in bacteria.</a> Journal of Biological Chemistry. 290 (1), 65-75 (2015).</li><li>Foit, L., George, J. S., Zhang, B. W., Brooks, C. L., Bardwell, J. C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone+activation+by+unfolding.">Chaperone activation by unfolding.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 1254-1262 (2013).</li><li>Sultana, S., Foti, A., Dahl, J. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+defense+systems+against+the+neutrophilic+oxidant+hypochlorous+acid.">Bacterial defense systems against the neutrophilic oxidant hypochlorous acid.</a> Infection and Immunity. 88 (7), 00964 (2020).</li><li>Dahl, J. -. U., Gray, M. J., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quality+control+under+oxidative+stress+conditions.">Protein quality control under oxidative stress conditions.</a> Journal of Molecular Biology. 427 (7), 1549-1563 (2015).</li><li>Groitl, B., Dahl, J. -. U., Schroeder, J. W., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+defense+systems+against+microbicidal+oxidants.">Pseudomonas aeruginosa defense systems against microbicidal oxidants.</a> Molecular Microbiology. 106 (3), 335-350 (2017).</li><li>Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+restriction+as+an+antimicrobial+function+of+fever.">Thermal restriction as an antimicrobial function of fever.</a> PLoS Pathogens. 12 (5), 1005577 (2016).</li><li>Richter, K., Haslbeck, M., Buchner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heat+shock+response:+life+on+the+verge+of+death.">The heat shock response: life on the verge of death.</a> Molecular Cell. 40 (2), 253-266 (2010).</li><li>Van Loi, V., Busche, T., Preu&#223;, T., Kalinowski, J., Bernhardt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AGXX+&#174;+antimicrobial+coating+causes+a+thiol-specific+oxidative+stress+response+and+protein+S-bacillithiolation+in+Staphylococcus+aureus.">The AGXX &#174; antimicrobial coating causes a thiol-specific oxidative stress response and protein S-bacillithiolation in Staphylococcus aureus.</a> Frontiers in Microbiology. 9, 3037 (2018).</li><li>Anfinsen, C. B., Scheraga, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+aspects+of+protein+folding.">Experimental and theoretical aspects of protein folding.</a> Advances in Protein Chemistry. 29, 205-300 (1975).</li><li>Schramm, F. D., Schroeder, K., Jonas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+in+bacteria.">Protein aggregation in bacteria.</a> FEMS Microbiology Reviews. 44 (1), 54-72 (2020).</li><li>Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+the+roles+of+chaperones+and+proteases+in+protein+folding+and+degradation+in+the+Escherichia+coli+cytosol.">Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol.</a> Molecular Microbiology. 40 (2), 397-413 (2001).</li><li>Gray, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphosphate+is+a+primordial+chaperone.">Polyphosphate is a primordial chaperone.</a> Molecular Cell. 53 (5), 689-699 (2014).</li><li>Weids, A. J., Ibstedt, S., Tam&#225;s, M. J., Grant, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stress+conditions+result+in+aggregation+of+proteins+with+similar+properties.">Distinct stress conditions result in aggregation of proteins with similar properties.</a> Scientific Reports. 6, 24554 (2016).</li><li>Mogk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+thermolabile+Escherichia+coli+proteins:+prevention+and+reversion+of+aggregation+by+DnaK+and+ClpB.">Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB.</a> EMBO Journal. 18 (24), 6934-6949 (1999).</li><li>Fay, A., Glickman, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+nonredundant+role+for+mycobacterial+DnaK+in+native+protein+folding.">An essential nonredundant role for mycobacterial DnaK in native protein folding.</a> PLoS Genetics. 10 (7), 1004516 (2014).</li><li>Schramm, F. D., Heinrich, K., Th&#252;ring, M., Bernhardt, J., Jonas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+regulatory+function+of+the+DnaK+chaperone+dictates+the+decision+between+proliferation+and+maintenance+in+Caulobacter+crescentus.">An essential regulatory function of the DnaK chaperone dictates the decision between proliferation and maintenance in Caulobacter crescentus.</a> PLoS Genetics. 13 (12), 1007148 (2017).</li><li>Maisonneuve, E., Fraysse, L., Moinier, D., Dukan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Existence+of+abnormal+protein+aggregates+in+healthy+Escherichia+coli+cells.">Existence of abnormal protein aggregates in healthy Escherichia coli cells.</a> Journal of Bacteriology. 190 (3), 887-893 (2008).</li><li>Heiss, A., Freisinger, B., Held-F&#246;hn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antibacterial+activity+of+silver-ruthenium+coated+hollow+microparticles.">Enhanced antibacterial activity of silver-ruthenium coated hollow microparticles.</a> Biointerphases. 12 (5), (2017).</li><li>Papnayotou, I., Sun, B., Roth, A. F., Davis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+induced+during+glass+bead+lysis+of+yeast.">Protein aggregation induced during glass bead lysis of yeast.</a> Yeast. 27 (10), 801-816 (2010).</li><li>Chuang, S. E., Blattner, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+twenty-six+new+heat+shock+genes+of+Escherichia+coli.">Characterization of twenty-six new heat shock genes of Escherichia coli.</a> Journal of Bacteriology. 175 (16), 5242-5252 (1993).</li><li>Imlay, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanisms+and+physiological+consequences+of+oxidative+stress:+Lessons+from+a+model+bacterium.">The molecular mechanisms and physiological consequences of oxidative stress: Lessons from a model bacterium.</a> Nature Reviews Microbiology. 11 (7), 443-454 (2013).</li><li>M&#252;hlhofer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heat+shock+response+in+yeast+maintains+protein+homeostasis+by+chaperoning+and+replenishing+proteins.">The heat shock response in yeast maintains protein homeostasis by chaperoning and replenishing proteins.</a> Cell Reports. 29 (13), 4593-4607 (2019).</li><li>Chandrangsu, P., Rensing, C., Helmann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+homeostasis+and+resistance+in+bacteria.">Metal homeostasis and resistance in bacteria.</a> Nature Reviews Microbiology. 15, 338-350 (2017).</li><li>Stevens, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP60/10+chaperonin+systems+are+inhibited+by+a+variety+of+approved+drugs,+natural+products,+and+known+bioactive+molecules.">HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules.</a> Bioorganic and Medicinal Chemistry Letters. 29 (9), 1106-1112 (2019).</li><li>Schramm, F. D., Schroeder, K., Alvelid, J., Testa, I., Jonas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-driven+displacement+of+protein+aggregates+along+the+cell+length+ensures+partitioning+to+both+daughter+cells+in+Caulobacter+crescentus.">Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus.</a> Molecular Microbiology. 111 (6), 1430-1448 (2019).</li></ol>

This protocol describes an optimized methodology for the analysis of protein aggregate formation after treatment of different E. coli strains with a proteotoxic antimicrobial. The protocol allows the simultaneous extraction of insoluble and soluble protein fractions from treated and untreated E. coli cells. Compared to existing protocols for protein aggregate isolation from cells14,15,16,<sup class="...

Bacteria are inevitably exposed to a myriad of environmental stresses, including low pH (e.g., in the mammalian stomach)1,2, reactive oxygen and chlorine species (ROS/RCS) (e.g., during oxidative burst in phagocytes)3,4,5, elevated temperatures (e.g., in hot springs or during heat-shock)6,7, and several potent antimicrobials (e.g., AGXX used in this protocol)8. Proteins are particularly vulnerable to any of these stressors, and exposure can provoke protein un-/misfolding that then seeds aggregation. All organisms employ protective systems that allow them to cope with protein misfolding9. However, severe stress can overwhelm the protein quality control machinery and disrupt the secondary and/or tertiary structure of proteins, which ultimately inactivates proteins. As a consequence, protein aggregates can severely impair critical cellular functions required for bacterial growth and survival, stress resistance, and virulence10. Therefore, research focusing on protein aggregation and its consequences in bacteria is a relevant topic due to its potential impact on infectious disease control.
Heat-induced protein unfolding and aggregation are often reversible7. In contrast, other proteotoxic stresses, such as oxidative stress, can cause irreversible protein modifications through the oxidation of specific amino acid side chains resulting in protein un-/misfolding and, eventually, protein aggregation4. Stress-induced formation of insoluble protein aggregates has been extensively studied in the context of molecular chaperones and their protective functions in yeast and bacteria11,12,13. Several protocols have been published that utilize a variety of biochemical techniques for the isolation and analysis of insoluble protein aggregates14,15,16,17. The existing protocols have mainly been used to study bacterial protein aggregation upon heat-shock and/or identification of molecular chaperones. While these protocols have certainly been an advancement to the field, there are some major inconveniences in the experimental procedures because they require (i) a large bacterial culture volume of up to 10 L14,17, (ii) complicated physical disruption processes, including the use of cell disruptors, French press, and/or sonication14,15,17, or (iii) time-consuming repeated washing and incubation steps15,16,17.
This paper describes a modified protocol that aims to address the limitations of the previous approaches and allows the analysis of the amount of protein aggregates formed in two different Escherichia coli strains after treatment with a proteotoxic antimicrobial surface coating. The coating is composed of metal-silver (Ag) and ruthenium (Ru)-conditioned with ascorbic acid, and its antimicrobial activity is achieved by the generation of reactive oxygen species8,18. Herein is a detailed description of the preparation of the bacterial culture after treatment with the antimicrobial compound and a comparison of protein aggregation status upon exposure of two E. coli strains with distinct susceptibility profiles to increasing concentration of the antimicrobial. The described method is inexpensive, fast, and reproducible and can be used to study protein aggregation in the presence of other proteotoxic compounds. In addition, the protocol can be modified to analyze the impact that specific gene deletions have on protein aggregation in a variety of different bacteria.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals/Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>67-64-1</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bisacrylamide solution 29:1</td>
			<td>Bio-Rad</td>
			<td>1610156</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Millipore Sigma</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Sigma</td>
			<td>E1014-5KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluestain 2 Protein ladder, 5-245 kDa</td>
			<td>GoldBio</td>
			<td>P008-500</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>GoldBio</td>
			<td>B-092-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>MP Biomedicals LLC</td>
			<td>821616</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sucrose</td>
			<td>Acros Organics</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetra acetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>SLBT9686</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic acid</td>
			<td>Millipore Sigma</td>
			<td>ARK2183-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>GoldBio</td>
			<td>G-630-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, ACS reagent</td>
			<td>Sigma-Aldrich</td>
			<td>320331-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (2-Propanol)</td>
			<td>Sigma</td>
			<td>402893-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Miller)</td>
			<td>Millipore Sigma</td>
			<td>L3522-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth with agar (Miller)</td>
			<td>Millipore Sigma</td>
			<td>L2897-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>GoldBio</td>
			<td>L-040-25</td>
			<td></td>
		</tr>
		<tr>
			<td>10x MOPS Buffer</td>
			<td>Teknova</td>
			<td>M2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>Thomas Scientific</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Millipore Sigma</td>
			<td>T9281-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma-Aldrich</td>
			<td>T4625-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Trichloroacetic acid</td>
			<td>Millipore Sigma</td>
			<td>T6399-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>GoldBio</td>
			<td>T-400-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL)</td>
			<td>Alkali Scientific</td>
			<td>JABG-1019</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask (125 mL)</td>
			<td>Carolina</td>
			<td>726686</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask (500 mL)</td>
			<td>Carolina</td>
			<td>726694</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer: -80 &#176;C</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads (0.5 mm)</td>
			<td>BioSpec Products</td>
			<td>1107-9105</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Hermle</td>
			<td>Z216MK</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentriguge tubes (1.7 mL)</td>
			<td>VWR International</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentriguge tubes (2.0 mL)</td>
			<td>Axygen Maxiclear Microtubes</td>
			<td>MCT-200-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cuvettes</td>
			<td>Fischer Scientific</td>
			<td>14-377-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>ThermoFisher Scientific</td>
			<td>EC105</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Alkali Scientific</td>
			<td>RS7235</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator (37 &#176;C)</td>
			<td>Benchmark Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small glass plate</td>
			<td>Bio-Rad</td>
			<td>1653311</td>
			<td></td>
		</tr>
		<tr>
			<td>Spacer plates (1 mm)</td>
			<td>Bio-Rad</td>
			<td>1653308</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermoscientific</td>
			<td>3339053</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge for 15 mL centrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical gel electrophoresis chamber</td>
			<td>Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher Vortex Genie 2</td>
			<td>12-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Benchmark Scientific</td>
			<td>H5000-HC</td>
			<td></td>
		</tr>
		<tr>
			<td>10 well comb</td>
			<td>Bio-Rad</td>
			<td>1653359</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and visualization of protein aggregates after treatment of escherichia coli with a proteotoxic stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62628/62628fig06.jpg" /> 
Figure 6: Representative results of antimicrobial-induced protein aggregation in commensal Escherichia coli strain MG1655 and UPEC strain CFT073. E. coli strains MG1655 and CFT073 were grown at 37 &#176;C and 300 rpm to OD600= 0.5-0.55 in MOPS-g media before they were treated with the indicated concentrations (-, 0 mg/mL; +, ...

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Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

Exposure to stress can cause protein aggregation and result in significant alterations in the phenotypic behavior of bacteria. The procedure described in this video allows the extraction and visualization of protein aggregates after stress treatment. In contrast to other published procedures, this protocol requires lower cell numbers and abstains from complicated physical disruption processes, as well as time-consuming washing and incubation steps.This protocol can be used to study protein aggregation in a wide variety of gram-negative and gram-positive bacteria. You can also analyze the effect of gene deletions or compare the efficacy of proteotoxic compounds. Begin by inoculating a single colony of E.coli strain MG1655 and uropathogenic E.coli strain CFT073, each into five milliliters of lysogeny broth, or LB, medium.Incubate the two broth cultures at 37 degrees Celsius and 300 rpm for 14 to 16 hours. Dilute each strain to an optical density at 600 nanometers, or OD600, of 0.1 into a 500-milliliter flask containing 70 milliliters of MOPS-g medium. Incubate the flasks at 37 degrees and 300 rpm until the mid-log phase is reached.After incubation, transfer 20 milliliters of each culture into three pre-warmed 125-milliliter flasks, and incubate them at 37 degrees Celsius and 300 rpm for two minutes. Prepare a two-milligram-per-milliliter antimicrobial compound solution in MOPS-g medium. Add this solution to each culture to reach the desired concentrations, and add the required volume of MOPS-g medium for the untreated control.Determine the OD600 of each culture after 45 minutes of stress treatment. For each sample, aliquot four milliliters of culture with an OD600 of one into 15-milliliter centrifuge tubes. Resuspend the cell pellets in 50 microliters of ice-cold lysis buffer.Incubate the samples for 30 minutes on ice. Add 360 microliters of ice-cold buffer A to the samples, and gently mix by pipetting. Transfer the samples into two-milliliter microcentrifuge tubes with around 200 microliters of 0.5-millimeter glass beads.Incubate the tubes for 30 minutes at eight degrees Celsius in a ThermoMixer with shaking at 1400 rpm. Incubate the tubes on ice for five minutes without shaking to settle the glass beads. Then transfer 200 microliters of the cell lysate into 1.7-milliliter microcentrifuge tubes.Centrifuge the cell lysate for 20 minutes at 16, 000 times g and four degrees Celsius. Collect the supernatant, which will be used as the soluble protein sample later. Use a pipette to resuspend the pellet in 200 microliters of ice-cold buffer A.Centrifuge of the tubes at 16, 000 times g and four degrees Celsius for 20 minutes.Then carefully remove the supernatant altogether. Use a pipette to carefully resuspend the pellet in 200 microliters of ice-cold buffer B.Centrifuge the tubes again, and carefully remove the supernatant. Then repeat the wash with buffer A.Resuspend the pellet in 100 microliters of 1x reducing SDS sample buffer, and boil it for five minutes in a ThermoMixer at 95 degrees Celsius.Mix one volume of 100%TCA with four volumes of the soluble protein sample collected earlier, and incubate for 10 minutes at four degrees Celsius for protein precipitation. Centrifuge the sample at 21, 000 times g and four degrees Celsius for five minutes to obtain the precipitate, and remove the supernatant. Use 200 microliters of ice-cold acetone to wash the pellet, to remove cellular debris.Centrifuge the sample again to obtain the precipitate, and remove the supernatant. To remove the remaining acetone from the pellets, keep the microcentrifuge tubes in the ThermoMixer at 37 degrees Celsius with their lids open. Then add 100 microliters of 1x reducing SDS buffer to completely dissolve the pellet, and boil the sample at 95 degrees Celsius for five minutes.Immediately load the sample on an SDS polyacrylamide gel for separation, or store the sample at minus 20 degrees Celsius to proceed later. Prepare two separating gels as described in the text manuscript. Pour the gel between the glass plates using a one-milliliter pipette, ensuring that the upper two centimeters are free of the mixture.Add 70%ethanol on top of the separating gel, creating an even interface between the two layers. Once the separating gels have polymerized, prepare the stacking gels using instructions in the text manuscript. Then remove the ethanol from the separating gels, and add the stacking gel solution.Carefully insert a comb with the desired number of pockets without introducing air bubbles, and allow the gel to polymerize for 20 to 30 minutes. Load four microliters of each sample, as well as the protein ladder, into separate wells. Then run the gel in tris-glycine running buffer at 144 volts for 45 minutes at room temperature.Use pre-warmed Fairbanks solution A to stain the gels on a rocker for 30 minutes and pre-warmed Fairbanks solution D to de-color the gels on a rocker until the desired background is achieved. An increase was observed in the amount of protein aggregate formed when cells were treated with 175 micrograms per milliliter and 200 micrograms per milliliter of the antimicrobial, as compared to untreated cells. The increase in the insoluble protein fraction was more pronounced in the more sensitive MG1655 strain.A decrease was observed in the amount of soluble proteins when cells were treated with 175 micrograms per milliliter and 200 micrograms per milliliter of the antimicrobial, as compared to untreated cells. When attempting this protocol, keep in mind that normalization to the optical density at 600 nanometers is important for comparison of the extent of protein aggregation in the samples.

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Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...