Name: 作者聚焦：探索疟疾和结核病感染中的热休克蛋白
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

这项工作得到了国际遗传工程和生物技术中心（ICGEB）资助号HDI/CRP/012，文达大学研究理事会，资助I595，科学与创新部（DSI）和南非国家研究基金会（NRF）的资助（资助号，75464和92598）授予AS。

1. 转型
注意：使用无菌玻璃器皿进行培养、移液器吸头以及新鲜制备和高压灭菌的培养基。在2x酵母胰蛋白胨（YT）[1.6%胰蛋白胨（w / v），1%酵母提取物（w / v），0.5%NaCl（w / v），1.5%琼脂（w / v）]琼脂中制备 大肠杆菌 细胞的培养物。协议中使用的一般试剂及其来源在 材料表中提供。
<ol><li>标记 2.0 mL 微量离心管并等分试样 50 μL 感受态大?...

使用 大肠杆菌 dnaK756 细胞评估 Hsp70 伴侣活性

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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. 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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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该方案证明了 大肠杆菌 dnaK756细胞在探索异源表达的 Hsp70 的伴侣功能方面的效用。该测定可用于筛选 纤维素中靶向 Hsp70 功能的抑制剂。然而，该方法的一个局限性是，在大 肠杆菌 中不能替代 DnaK 的 Hsp70 与该测定不兼容。一些非天然 Hsp70 缺乏翻译后修饰21 可能是它们在 大肠杆菌 系统中缺乏功能的原因。基于酵母的互补测定22可以?...

热休克蛋白通过促进蛋白质折叠、防止蛋白质聚集和逆转蛋白质错误折叠，发挥着分子伴侣的重要作用 1,2。热休克蛋白 70 （Hsp70） 是最突出的分子伴侣之一，在蛋白质稳态中起着核心作用 3,4。DnaK 是大肠杆菌 Hsp70 同源物5。
已经开发了各种生物物理、生化和基于细胞的测定法来探索 Hsp70 的伴侣活性并筛选靶向该伴侣的抑制剂 6,7,8。Hsp70 是一种高度保守的蛋白质。出于这个原因，据报道，几种真核生物的 Hsp70，例如恶性疟原虫（疟疾的主要病原体）可替代大肠杆菌中的 DnaK 功能 6,9。通过这种方式，开发了一种基于大肠杆菌的互补测定法，涉及大肠杆菌中Hsp70s的异源表达，以探索其细胞保护功能。通常，该测定涉及利用缺乏 DnaK 或表达功能受损的天然 DnaK 的大肠杆菌细胞。虽然DnaK在正常条件下对大肠杆菌的生长不是必需的，但当细胞在高温或其他形式的压力等压力条件下生长时，它变得至关重要10,11。
使用互补测定法研究 Hsp70 功能的大肠杆菌菌株包括大肠杆菌 dnaK103 （BB2393 [C600 dnaK103（Am） thr：：Tn10]） 和大肠杆菌 dnaK756。大肠杆菌 dnaK103 细胞产生无功能的截短 DnaK，因此，细胞在 30 °C 下充分生长，而菌株对冷和热应激敏感12,13。同样，大肠杆菌 dnaK756/BB2362 （dnaK756 recA：：TcR Pdm1,1） 菌株不会在 40 °C以上生长 14,15。大肠杆菌 dnaK756 菌株表达突变的天然 DnaK （DnaK756），其特征是在 32、455 和 468 位点有三种甘氨酸到天冬氨酸的取代，导致蛋白稳态结果受损。因此，该菌株对噬菌体λ DNA14具有抗性。此外，大肠杆菌 dnaK756 表现出升高的 ATP 酶活性，而其对核苷酸交换因子 GrpE 的亲和力降低16。大肠杆菌DnaK突变菌株可作为通过互补方法研究Hsp70伴侣活性的理想模型。由于 DnaK 仅在压力条件下是必需的，因此互补测定通常在高温下进行（图 1）。使用大肠杆菌进行这项研究的一些优点包括其表征良好的基因组、快速生长以及培养和维护的低成本17。
在本文中，我们详细描述了一种涉及使用大肠杆菌 dnaK756 细胞来研究 Hsp70 功能的方案。我们在测定中使用的 Hsp70 是野生型 DnaK 及其嵌合衍生物 KPf（由 DnaK 的 ATP 酶结构域与恶性疟原虫 Hsp70-1 6,18 的 C 端底物结合结构域融合组成）。KPf-V436F 被异源表达为阴性对照，因为该突变基本上阻止其结合底物，从而消除其伴侣活性9。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>8,05,740</td>
			<td>Constituent for sample loading dye</td>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

escherichia coli -based complementation assay to study the chaperone function of heat shock protein 70

热休克蛋白 70 （Hsp70） 是一种保守的蛋白质，可促进细胞内其他蛋白质的折叠，使其成为分子伴侣。虽然 Hsp70 对于在正常条件下生长的大 肠杆菌 细胞不是必需的，但这种伴侣对于在高温下生长是必不可少的。由于 Hsp70 是高度保守的，因此研究来自不同物种的 Hsp70 基因伴侣功能的一种方法是在大肠杆菌菌株中异源表达它们，这些 菌 株要么缺乏 Hsp70，要么表达功能受损的天然 Hsp70。 大肠杆菌 dnaK756 细胞不能支持噬菌体 DNA。此外，它们的天然 Hsp70 （DnaK） 表现出升高的 ATP 酶活性，同时表现出对 GrpE（Hsp70 核苷酸交换因子）的亲和力降低。因此， 大肠杆菌 dnaK756 细胞在 30 °C 至 37 °C 的温度范围内充分生长，但它们在高温 （>40 °C） 下死亡。出于这个原因，这些细胞可作为研究 Hsp70 伴侣活性的模型。在这里，我们描述了应用这些细胞进行互补测定的详细方案，从而能够研究 Hsp70 的纤维素 伴侣功能。

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.

作者聚焦：探索疟疾和结核病感染中的热休克蛋白

基于大肠杆菌的互补测定研究热休克蛋白的伴侣功能 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.

图2显示了分别在37°C和43.5°C的允许生长温度下发现和培养的含有细胞的扫描琼脂的图像。在图 2 的右侧，切除的蛋白质印迹成分代表大肠杆菌 dnaK756 细胞中 DnaK、KPf 和 KPf-V436F 的表达。正如预期的那样，在37°C的允许生长温度下培养的所有大肠杆菌dnaK756细胞都成功生长。然而，在43.5°C的非允许生长条件下，只有异源表达DnaK和KPf的细胞能?...

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

该方案证明了热休克蛋白70（Hsp70）的伴侣活性。 大肠杆菌 dnaK756 细胞可作为测定的模型，因为它们含有天然的、功能受损的 Hsp70，使其容易受到热应激的影响。功能性 Hsp70 的异源引入挽救了细胞的生长缺陷。

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

我研究了热休克蛋白在为引起人类感染（如疟疾和结核病）的抗原提供未来静态保护方面的作用。我的研究主要集中在这些引起人类感染的病原体的热休克蛋白机制的结构和功能特征上。中暑蛋白通常具有高度的概念性，但是，它们表现出一定程度的功能特化。此外，它们还与耐药性有关。因此，目前正在努力使它们在各种疾病模型中逆转耐药性。它们包括生物化学、生物物理、生物信息学和细胞生物学技术。换句话说，各种领域的技术被用来研究这些蛋白质的作用。在我们的案例中，对疟原虫蛋白质的研究是疟疾的主要病原体，它们难以生产，这限制了研究这些蛋白质的能力。例如，很难产生足够的水平来结晶和成像蛋白质。我们已经在疟疾寄生虫中建立了这些蛋白质的一些功能网络。我们还建立了检测方法，以研究在引起疟疾和结核病的病原体中靶向这些蛋白质的化合物。本方案以等同为模型，探讨HSP 70的伴侣蛋白折叠作用和细胞保护功能。该方案也可用于筛选靶向蛋白70的小分子抑制剂。

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.