transformation of e. coli cells for a complementation assay to assess hsp70 chaperone activity

Avaliação da atividade da chaperona Hsp70 usando células Escherichia coli dnaK756

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.

Autor em destaque: Explorando proteínas de choque térmico em infecções por malária e tuberculose

Ensaio de Complementação Baseado em Escherichia coli para Estudo da Função Chaperone da Proteína de Choque Térmico 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.

Transformação de células de E. coli para um ensaio de complementação para avaliar a atividade de chaperona Hsp70

Para começar, rotule os tubos de microcentrífuga de dois mililitros. Pipetar alíquotas de 50 microlitros de culturas de E.coli DnaK756 para dentro dos tubos. Coloque os tubos sobre o gelo.Adicionar 10 a 50 nanogramas de DNA plasmidial em cada tubo separado com as alíquotas de E.coli. Mantenha os tubos no gelo por 30 minutos. Em seguida, coloque as células a 42 graus Celsius por 60 segundos para aquecer as células.Devolva os tubos ao gelo por 10 minutos. Pipetar 950 microlitros de caldo de triptona fresco duas vezes para dentro dos tubos e, em seguida, incubar os tubos em um agitador a 37 graus Celsius. Pipetar 100 microlitros da cultura celular transformada e espalhar em placas de ágar com duas vezes triptona de levedura contendo antibióticos.Centrifugar o resto das células durante um minuto a 5.000 G a quatro graus Celsius. Decantar cerca de 800 microlitros do caldo. Ressuspender as células peletizadas no meio restante.Coloque as células recuperadas em uma placa de ágar triptona duas vezes levedura e, em seguida, incube ambas as placas de ágar a 37 graus Celsius durante a noite. Para plaquear as células, use um laço estéril para pegar uma única colônia dos transformadores. Inocular em 10 mililitros de duas vezes o caldo de triptona de levedura, suplementado com antibióticos.Agora, prepare diluições seriadas das células de 100 a 10 até o quinto negativo em tubos de microcentrífuga de dois mililitros. Antes de detectar as células, coloque placas de ágar suplementadas com antibióticos e IPTG em um forno a 40 graus Celsius com suas tampas parcialmente abertas. Quando as placas estiverem secas o suficiente, identifique dois microlitros das células diluídas em série nas placas de ágar.Coloque cada amostra em duas placas separadas. Incubar uma placa à temperatura de crescimento permissiva de 37 graus Celsius e a outra à temperatura de crescimento não permissiva de 43,5 graus Celsius. Para confirmar a expressão de proteínas recombinantes, use uma alça estéril para pegar parte das células restantes da mesma colônia de transformantes.Inocular as células em 10 mililitros de fermento antibiótico suplementado com caldo de triptona. Incube as culturas em um agitador durante a noite a 37 graus Celsius. Todas as células de E.coli DnaK756 cresceram à temperatura de crescimento permissiva de 37 graus Celsius.No entanto, apenas células heterólogas expressando DnaK e KPf cresceram na temperatura de crescimento não permissiva. As células mutantes que expressam KPf-V436F só cresceram a 37 graus Celsius, mas não conseguiram crescer a 43,5 graus Celsius.

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Transformation of E. coli Cells for a Complementation Assay to Assess Hsp70 Chaperone Activity

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