Name: Forfatter i søkelyset: Utforsking av varmesjokkproteiner i malaria- og tuberkuloseinfeksjoner
Uploaded: 2024-03-08T14:25:00
Description: Watch this Scientific Journal Video about Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections at JoVE.com

Arbeidet ble støttet med tilskuddsmidler hentet fra International Centre for Genetic Engineering and Biotechnology (ICGEB) grant number, HDI/CRP/012, Research Directorate ved University of Venda, grant I595, Department of Science and Innovation (DSI) og National Research Foundation (NRF) i Sør-Afrika (grant numbers, 75464 &; 92598) tildelt AS.

1. Transformasjon
MERK: Bruk sterilt glass til kultur, pipettespisser og nytilberedte og autoklaverte medier. Forbered kulturer av E. coli-cellene i 2x gjærtrypton (YT) [1,6% trypton (w / v), 1% gjærekstrakt (w / v), 0,5% NaCl (w / v), 1,5% agar (w / v)] agar. Generelle reagenser som brukes i protokollen og deres kilder er gitt i materialfortegnelsen.
<ol><li>Merk 2,0 ml mikrosentrifugerør og alikot 50 μL kompetente E. coli dnaK756-celler</e...

Vurdering av Hsp70 Chaperon-aktivitet ved bruk av Escherichia coli dnaK756-celler

<ol>
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Immunol. 58 (1), 61-74 (2010).</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 J. 18 (24), 6934-6949 (1999).</li><li>Edkins, A. L., Boshoff, A., Shonhai, A., Picard, D., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+structural+and+functional+features+of+molecular+chaperones.+Heat+shock+proteins+of+malaria.">General structural and functional features of molecular chaperones. Heat shock proteins of malaria.</a> Adv Exp Med Biol. , (2021).</li><li>Bertelsen, E. B., Chang, L., Gestwicki, J. E., Zuiderweg, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+conformation+of+wild-type+E.+coli.+Hsp70+(DnaK)+chaperone+complexed+with+ADP+and+substrate.">Solution conformation of wild-type E. coli. Hsp70 (DnaK) chaperone complexed with ADP and substrate.</a> PNAS. 106 (21), 8471-8476 (2009).</li><li>Shonhai, A., Boshoff, A., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+heat+shock+protein+70+is+able+to+suppress+the+thermosensitivity+of+an+Escherichia+coli+DnaK+mutant+strain.">Plasmodium falciparum heat shock protein 70 is able to suppress the thermosensitivity of an Escherichia coli DnaK mutant strain.</a> Mol Genet Genomics. 274, 70-78 (2005).</li><li>Shonhai, A., Botha, M., de Beer, T. A., Boshoff, A., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+study+of+a+Plasmodium+falciparum+Hsp70+using+three-dimensional+modelling+and+in+vitro+analyses.">Structure-function study of a Plasmodium falciparum Hsp70 using three-dimensional modelling and in vitro analyses.</a> Protein Pept Lett. 15 (10), 1117-1125 (2008).</li><li>Cockburn, I. L., Boshoff, A., Pesce, E. -. R., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+modulation+of+plasmodial+Hsp70s+by+small+molecules+with+antimalarial+activity.">Selective modulation of plasmodial Hsp70s by small molecules with antimalarial activity.</a> Biol Chem. 395 (11), 1353-1362 (2014).</li><li>Makhoba, X. H., 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=Use+of+a+chimeric+Hsp70+to+enhance+the+quality+of+recombinant+Plasmodium+falciparum+s-adenosylmethionine+decarboxylase+protein+produced+in+Escherichia+coli.">Use of a chimeric Hsp70 to enhance the quality of recombinant Plasmodium falciparum s-adenosylmethionine decarboxylase protein produced in Escherichia coli.</a> PLoS One. 11 (3), 0152626 (2016).</li><li>Bukau, B., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+defects+caused+by+deletion+of+the+Escherichia+coli+dnaK+gene+indicate+roles+for+heat+shock+protein+in+normal+metabolism.">Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.</a> J Bact. 171 (5), 2337-2346 (1989).</li><li>Makumire, S., Revaprasadu, N., Shonhai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK+protein+alleviates+toxicity+induced+by+citrate-coated+gold+nanoparticles+in+Escherichia+coli.">DnaK protein alleviates toxicity induced by citrate-coated gold nanoparticles in Escherichia coli.</a> PLoS One. 10 (4), 0121243 (2015).</li><li>Spence, J., Cegielska, A., Georgopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Escherichia+coli+heat+shock+proteins+DnaK+and+HtpG+(C62.+5)+in+response+to+nutritional+deprivation.">Role of Escherichia coli heat shock proteins DnaK and HtpG (C62. 5) in response to nutritional deprivation.</a> J Bact. 172 (12), 7157-7166 (1990).</li><li>Mayer, M. P., 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=Multistep+mechanism+of+substrate+binding+determines+chaperone+activity+of+Hsp70.">Multistep mechanism of substrate binding determines chaperone activity of Hsp70.</a> Nat Struct Biol. 7 (7), 586-593 (2000).</li><li>Georgopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+bacterial+gene+(groP+C)+which+affects+&#955;+DNA+replication.">A new bacterial gene (groP C) which affects &#955; DNA replication.</a> Mol Genet Genomics. 151 (1), 35-39 (1977).</li><li>Tilly, K., McKittrick, N., Zylicz, M., Georgopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dnaK+protein+modulates+the+heat-shock+response+of+Escherichia+coli.">The dnaK protein modulates the heat-shock response of Escherichia coli.</a> Cell. 34 (2), 641-646 (1983).</li><li>Buchberger, A., Gassler, C. S., Buttner, M., McMacken, R., 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=Functional+defects+of+the+DnaK756+mutant+chaperone+of+Escherichia+coli+indicate+distinct+roles+for+amino-and+carboxyl-terminal+residues+in+substrate+and+co-chaperone+interaction+and+interdomain+communication.">Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino-and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication.</a> J Biol Chem. 274 (53), 38017-38026 (1999).</li><li>Taj, M. K., 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=Escherichia+coli+as+a+model+organism.">Escherichia coli as a model organism.</a> Int J Eng Res. 3 (2), 1-8 (2014).</li><li>Sato, S., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+cochaperonins+in+apicomplexan+parasites.">Organelle-specific cochaperonins in apicomplexan parasites.</a> Mol Biochem Parasitol. 141 (2), 133-143 (2005).</li><li>Molecular characterisation of the chaperone properties of Plasmodium falciparum. heat shock protein 70. Rhodes University Available from: <a target="_blank" href="https://commons.ru.ac.za/vital/access/manager/Repository/vital:3977?site_name=Rhodes+University">https://commons.ru.ac.za/vital/access/manager/Repository/vital:3977?site_name=Rhodes+University</a> (2007)</li><li>Makumire, S., 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=Mutation+of+GGMP+repeat+segments+of+Plasmodium+falciparum+Hsp70-1+compromises+chaperone+function+and+Hop+co-chaperone+binding.">Mutation of GGMP repeat segments of Plasmodium falciparum Hsp70-1 compromises chaperone function and Hop co-chaperone binding.</a> Int J Mol Sci. 22 (4), 2226 (2021).</li><li>Nitika, P. C. M., Truman, A. W., Truttmann, M. 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Protokollen demonstrerer nytten av E. coli dnaK756-celleri å utforske chaperone-funksjonen til heterologisk uttrykt Hsp70. Denne analysen kan brukes til screenhemmere rettet mot Hsp70-funksjonen i cellulo. Imidlertid er en begrensning av denne metoden at Hsp70s ikke kan erstatte DnaK i E. coli ikke er kompatible med denne analysen. Mangel på posttranslasjonell modifikasjon21 av noen ikke-innfødte Hsp70s kan forklare deres mangel på funksjon innenfor E. coli-syste...

Varmesjokkproteiner spiller en viktig rolle som molekylære chaperoner ved å lette proteinfolding, forhindre proteinaggregering og reversere proteinfolding 1,2. Varmesjokkprotein 70 (Hsp70) er en av de mest fremtredende molekylære chaperonene, og spiller en sentral rolle i proteinhomeostase 3,4. DnaK er E. coli Hsp70 homolog5.
Ulike biofysiske, biokjemiske og cellebaserte analyser er utviklet for å utforske chaperonaktiviteten til Hsp70 og for å skjerme for hemmere rettet mot denne chaperone 6,7,8. Hsp70 er et svært konservert protein. Av denne grunn har flere Hsp70-er av eukaryote organismer, som Plasmodium falciparum (hovedmidlet til malaria), blitt rapportert å erstatte DnaK-funksjonen i E. coli 6,9. På denne måten har en E. coli-basert komplementeringsanalyse blitt utviklet som involverer det heterologe uttrykket av Hsp70s i E. coli for å utforske deres cytoprotektive funksjon. Vanligvis innebærer denne analysen bruk av E. coli-celler som enten er mangelfull for DnaK eller som uttrykker en innfødt DnaK som er funksjonelt kompromittert. Mens DnaK ikke er avgjørende for E. coli-vekst under normale forhold, blir det viktig når cellene dyrkes under stressende forhold som forhøyede temperaturer eller andre former for stress10,11.
E. coli-stammer som er utviklet for å studere Hsp70-funksjonen ved hjelp av en komplementeringsanalyse, inkluderer E. coli dnaK103 (BB2393 [C600 dnaK103 (Am) thr:: Tn10]) og E. coli dnaK756. E. coli dnaK103-celler produserer en avkortet DnaK som ikke er funksjonell, og som sådan vokser cellene tilstrekkelig ved 30 °C, mens belastningen er følsom for kulde og varmespenning 12,13. På samme måte vokser ikke E. coli dnaK756/BB2362 (dnaK756 recA::TcR Pdm1,1)-stammen over 40 °C14,15. E. coli dnaK756-stammen uttrykker en mutant innfødt DnaK (DnaK756) karakterisert ved tre glycin-til-aspartat-substitusjoner i posisjonene 32, 455 og 468, noe som gir opphav til kompromitterte proteostatiske utfall. Følgelig er denne stammen resistent mot bakteriofag λ DNA14. I tillegg viser E. coli dnaK756 forhøyet ATPase-aktivitet, mens affiniteten til nukleotidutvekslingsfaktoren, GrpE, reduseres16. E. coli DnaK-mutantstammer fungerer som ideelle modeller for å undersøke chaperonaktiviteten til Hsp70 gjennom en komplementeringstilnærming. Siden DnaK bare er essensielt under stressende forhold, utføres komplementeringsanalysen vanligvis ved forhøyede temperaturer (figur 1). Noen fordeler med å bruke E. coli for denne studien inkluderer det velkarakteriserte genomet, rask vekst og de lave kostnadene ved dyrking og vedlikehold17.
I denne artikkelen beskriver vi i detalj en protokoll som involverer bruk av E. coli dnaK756-celler for å studere funksjonen til Hsp70. Hsp70-ene vi benyttet i analysen er villtype DnaK og dets kimære derivat, KPf (består av ATPase-domenet til DnaK smeltet sammen med det C-terminale substratbindende domenet til Plasmodium falciparum Hsp70-1 6,18). KPf-V436F ble heterologisk uttrykt som en negativ kontroll siden mutasjonen i hovedsak blokkerer den fra bindende substrater, og dermed opphever sin chaperoneaktivitet9.

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			<td>2-&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>8,05,740</td>
			<td>Constituent for sample loading dye</td>
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		<tr>
			<td>Acetic acid</td>
			<td>Labchem</td>
			<td>101005125</td>
			<td>Constituent of destainer</td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>8008300100</td>
			<td>Component of SDS</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Merck</td>
			<td>HG000BX1.500</td>
			<td>Constituent of medium and liquid growth assay</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Clever Scientific</td>
			<td>14131031</td>
			<td>Certified molecular biology agarose</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>101875295</td>
			<td>Constituent for SDS-PAGE gel</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>VWR International</td>
			<td>0339&#8212;EU&#8212;25G</td>
			<td>Selective antibiotic</td>
		</tr>
		<tr>
			<td>Bis</td>
			<td>Sigma-aldrich</td>
			<td>1015460100</td>
			<td>Component of SDS</td>
		</tr>
		<tr>
			<td>Bromophenol</td>
			<td>Sigma-Aldrich</td>
			<td>0449-25G</td>
			<td>Constituent for sample loading dye</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td>For competent cells preparation</td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue</td>
			<td>VWR International</td>
			<td>443293X</td>
			<td>SDS-PAGE dye</td>
		</tr>
		<tr>
			<td>Dibasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>RB10368</td>
			<td>Constituent of PBS buffer</td>
		</tr>
		<tr>
			<td>ECL</td>
			<td>Thermofischer Scientific</td>
			<td>32109</td>
			<td>Western blot detection reagent</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Thermofischer Scientific</td>
			<td>17898</td>
			<td>DNA intercalating dye</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Merck</td>
			<td>SAAR2676520L</td>
			<td>Constituent for sample loading dye</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>VWR International</td>
			<td>10119CU</td>
			<td>Component of SDS</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Glentham life sciences</td>
			<td>162IL</td>
			<td>inducer</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Melford</td>
			<td>K0126</td>
			<td>Selective antibiotic</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Merck</td>
			<td>SAAR4123000EM</td>
			<td>Constituent of medium and liquid growth assay</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Labchem</td>
			<td>113140129</td>
			<td>Constituent of destainer</td>
		</tr>
		<tr>
			<td>Monobasic potassium phosphate</td>
			<td>Merck</td>
			<td>1,04,87,30,250</td>
			<td>Constituent of PBS buffer</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck</td>
			<td>HG000BX4.250</td>
			<td>Constituent of medium and liquid growth assay</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>SAAR5042020EM</td>
			<td>Constituent of PBS buffer</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Thermofischer scientific</td>
			<td>PB7320</td>
			<td>Western blot membrane</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck</td>
			<td>SAAR5822320EM</td>
			<td>Constituent of medium and liquid growth assay</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate</td>
			<td>VWR International</td>
			<td>108073</td>
			<td>To resolve expressed proteins</td>
		</tr>
		<tr>
			<td>Spectramax iD3</td>
			<td>Separations</td>
			<td>373705019</td>
			<td>Automated plate reader</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>VWR international</td>
			<td>ACRO420580500</td>
			<td>Component of SDS gel</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Duchefa Biochemies</td>
			<td>T0150.0025</td>
			<td>Selective antibiotic</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR International</td>
			<td>19A094101</td>
			<td>Component of SDS gel</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Merck</td>
			<td>SAAR3164500XF</td>
			<td>Constituent for Western wash buffer</td>
		</tr>
		<tr>
			<td>Western transfer chamber</td>
			<td>Thermofisher Scientific</td>
			<td>PB0112</td>
			<td>Transfer of protein to nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Merck</td>
			<td>HG000BX6.500</td>
			<td>Constituent of medium and liquid growth assay</td>
		</tr>
		<tr>
			<td>&#945;-DnaK antibody</td>
			<td>Inqaba</td>
			<td>BK CAC09317</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>&#945;-rabbit antibody</td>
			<td>Thermofischer scientific</td>
			<td>31460</td>
			<td>Secondary antibody</td>
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escherichia coli -based complementation assay to study the chaperone function of heat shock protein 70

Varmesjokkprotein 70 (Hsp70) er et konservert protein som letter foldingen av andre proteiner i cellen, noe som gjør det til en molekylær chaperone. Mens Hsp70 ikke er avgjørende for E. coli-celler som vokser under normale forhold, blir denne chaperone uunnværlig for vekst ved forhøyede temperaturer. Siden Hsp70 er svært konservert, er en måte å studere chaperone-funksjonen til Hsp70-gener fra forskjellige arter å heterologisk uttrykke dem i E. coli-stammer som enten er mangelfulle i Hsp70 eller uttrykker en innfødt Hsp70 som er funksjonelt kompromittert. E. coli dnaK756 celler er ikke i stand til å støtte λ bakteriofag DNA. Videre viser deres opprinnelige Hsp70 (DnaK) forhøyet ATPase-aktivitet mens de demonstrerer redusert affinitet for GrpE (Hsp70 nukleotidutvekslingsfaktor). Som et resultat vokser E. coli dnaK756-celler tilstrekkelig ved temperaturer fra 30 ° C til 37 ° C, men de dør ved forhøyede temperaturer (>40 ° C). Av denne grunn tjener disse cellene som en modell for å studere chaperonaktiviteten til Hsp70. Her beskriver vi en detaljert protokoll for anvendelse av disse cellene for å utføre en komplementeringsanalyse, noe som muliggjør studiet av cellulo-chaperonfunksjonen til Hsp70.

Heat shock proteins play an important role as molecular chaperones by facilitating protein folding, preventing protein aggregation, and reversing protein misfolding1,2. Heat shock protein 70 (Hsp70) is one of the most prominent molecular chaperones, playing a central role in protein homeostasis3,4. DnaK is the E. coli Hsp70 homologue5.
Various biophysical, biochemical, and cell-based assays have been developed to explore the chaperone activity of Hsp70 and to screen for inhibitors targeting this chaperone6,7,8. Hsp70 is a highly conserved protein. For this reason, several Hsp70s of eukaryotic organisms, such as Plasmodium falciparum (the main agent of malaria), have been reported to substitute for DnaK function in E. coli6,9. In this way, an E. coli-based complementation assay has been developed involving the heterologous expression of Hsp70s in E. coli to explore their cytoprotective function. Typically, this assay involves the utilization of E. coli cells that are either deficient for DnaK or that express a native DnaK that is functionally compromised. While DnaK is not essential for E. coli growth under normal conditions, it becomes essential when the cells are grown under stressful conditions such as elevated temperatures or other forms of stress10,11.
E. coli strains that have been developed to study Hsp70 function using a complementation assay include E. coli dnaK103&#160;(BB2393 [C600 dnaK103(Am) thr::Tn10]) and E. coli dnaK756. E. coli dnaK103&#160;cells produce a truncated DnaK that is non-functional, and as such, the cells grow adequately at 30 &#176;C, while the strain is sensitive to cold and heat stress12,13. Similarly, the E. coli dnaK756/BB2362 (dnaK756&#160;recA::TcR Pdm1,1) strain does not grow above 40 &#176;C14,15. The E. coli dnaK756&#160;strain expresses a mutant native DnaK (DnaK756) characterized by three glycine-to-aspartate substitutions at positions 32, 455, and 468, giving rise to compromised proteostatic outcomes. Consequently, this strain is resistant to bacteriophage &#955; DNA14. Additionally, E. coli&#160;dnaK756 exhibits elevated ATPase activity, while its affinity for the nucleotide exchange factor, GrpE, is reduced16. E. coli DnaK mutant strains serve as ideal models for investigating the chaperone activity of Hsp70 through a complementation approach. Since DnaK is only essential under stressful conditions, the complementation assay is typically conducted at elevated temperatures (Figure 1). Some advantages of using E. coli for this study include its well-characterized genome, rapid growth, and the low cost of culturing and maintenance17.
In this article, we describe in detail a protocol involving the use of E. coli dnaK756&#160;cells to study the function of Hsp70. The Hsp70s we employed in the assay are wild-type DnaK and its chimeric derivative, KPf (made up of the ATPase domain of DnaK fused to the C-terminal substrate-binding domain of Plasmodium falciparum Hsp70-16,18). KPf-V436F was heterologously expressed as a negative control since the mutation essentially blocks it from binding substrates, thus abrogating its chaperone activity9.

Forfatter i søkelyset: Utforsking av varmesjokkproteiner i malaria- og tuberkuloseinfeksjoner

Escherichia coli -basert komplementeringsanalyse for å studere chaperone-funksjonen til varmesjokkprotein 70

I study the role of heat shock proteins in conferring future static protection to antigens that cause human infections such as malaria and tuberculosis. My research essentially focuses on the structure and function features of the heat shock protein machinery of these pathogens that cause human infections. Heatstroke proteins are generally highly concept, however, they display some level of functional specialization.In addition, they are implicated in drug resistant. As such, there are ongoing efforts to target them towards reversing drug resistance in various disease models. They include biochemical, biophysical, bioinformatics, and cell biologic techniques.In other words, various areas of techniques are used to study the roles of these proteins. In our case, the study of proteins of Plasmodium was the main agent of malaria and that they are difficult to produce and this limits the capability to study these proteins. For example, it is difficult to generate sufficient levels to crystallize and image the proteins.We have established some of the functional networks of these proteins in the malaria parasite. We have also established assays to study the compounds targeting these proteins in agents causing malaria and TB.The current protocol is used to explore the chaperone protein folding role and then cytoprotective function of HSP 70 using equal as a model. The protocol could also be adopted for screening small molecule inhibitors targeting protein 70.

Figur 2 viser et bilde av den skannede agar med celler som ble oppdaget og dyrket ved den tillatte veksttemperaturen på henholdsvis 37 °C og 43,5 °C. På høyre side av figur 2 representerer utskårne vestlige blotkomponenter uttrykket av DnaK, KPf og KPf-V436F i E. coli dnaK756-celler. Som forventet klarte alle E. coli dnaK756-cellene dyrket ved den tillatte veksttemperaturen på 37 °C å vokse. Under de ikke-tillatte vekstbetingelsene på...

Watch this Scientific Journal Video about Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections at JoVE.com

Denne protokollen demonstrerer chaperoneaktiviteten til varmesjokkprotein 70 (Hsp70). E. coli dnaK756-celler tjener som en modell for analysen da de har en innfødt, funksjonshemmet Hsp70, noe som gjør dem utsatt for varmestress. Den heterologe introduksjonen av funksjonell Hsp70 redder vekstmangelen til cellene.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

Jeg studerer rollen til varmesjokkproteiner i å gi fremtidig statisk beskyttelse til antigener som forårsaker menneskelige infeksjoner som malaria og tuberkulose. Min forskning fokuserer i hovedsak på strukturen og funksjonsfunksjonene til varmesjokkproteinmaskineriet til disse patogenene som forårsaker menneskelige infeksjoner. Heteslagproteiner er generelt svært konsept, men de viser et visst nivå av funksjonell spesialisering.I tillegg er de involvert i stoffresistent. Som sådan er det pågående arbeid for å målrette dem mot å reversere stoffresistens i ulike sykdomsmodeller. De inkluderer biokjemiske, biofysiske, bioinformatikk og cellebiologiske teknikker.Med andre ord brukes ulike områder av teknikker for å studere rollene til disse proteinene. I vårt tilfelle var studien av proteiner av Plasmodium hovedagenten for malaria og at de er vanskelige å produsere, og dette begrenser evnen til å studere disse proteinene. For eksempel er det vanskelig å generere tilstrekkelige nivåer for å krystallisere og avbilde proteinene.Vi har etablert noen av de funksjonelle nettverkene av disse proteinene i malariaparasitten. Vi har også etablert analyser for å studere forbindelsene som retter seg mot disse proteinene i midler som forårsaker malaria og TB. Den nåværende protokollen brukes til å utforske chaperoneproteinfoldingsrollen og deretter cytoprotektiv funksjon av HSP 70 ved å bruke lik som modell. Protokollen kan også vedtas for screening av småmolekylære hemmere rettet mot protein 70.

escherichia-coli-based-complementation-assay-to-study-chaperone

Research

JoVE Journal

Biochemistry

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.