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Order Martellivirales: Virgaviridae
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Developing further the E. coli expression of the TMV coat gene, Hwang et al. (1998) found that both GroEL and DnaK chaperons had significant direct or indirect effects on the overall expression, stability, folding, and assembly of the TMV coat protein in vivo. The overproduction of GroEL or GroES alone had little effect. However, cooverexpression of GroEL and GroES resulted in a twofold increase in soluble TMV coat and a fourfold rise in the assembled TMV-like particles in vivo. Moreover, TMV coat was shown to interact directly with GroEL in vivo (Hwang et al. 1998). Later, Dedeo et al. (2010) constructed and expressed in E. coli a “circular permutant” of TMV coat, where the N- and C-termini were reengineered, and a short loop was placed to close the sequence gap, as described later in detail.
The Stress Response and Stress Proteins
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Martin E. Feder, Dawn A. Parsell, Susan L. Lindquist
Numerous studies have now demonstrated that some stress proteins can contribute to the refolding and reactivation of denatured proteins in vitro. A typical experiment involves denaturing a test protein (e.g., rhodanese, dihydrofolate reductase, luciferase, citrate synthase) with a chemical denaturant (e.g., urea, guanidine) or heat, removing the denaturant, and characterizing either the structure or the function of the test protein in the presence or absence of presumptive chaperones and their partner proteins. GroEL and GroES (the E. coli counterparts of hsp60 and hsp10, respectively) in combination have now repeatedly been shown to chaperone refolding and reactivation of a variety of test proteins (e.g., Figure 4); this activity requires ATP. To date, only two such studies have documented similar activity for DnaK (the E. coli counterpart of hsp70) and its partner proteins DnaJ and GrpE. In one study, reactivation of RNA polymerase required a 60-fold molar excess of DnaK.25 The other study26 examined firefly luciferase expressed in E. coli and showed that less excessive quantities of DnaJ, DnaK, and GrpE in combination could prevent the aggregation of heat-denatured luciferase and eventually release refolded luciferase; again, this activity requires ATP.
A Mycobacterial 65 kD Heat-Shock Protein and T Cells in Experimental Arthritis
Published in Thomas F. Kresina, Monoclonal Antibodies, Cytokines, and Arthritis, 2020
Willem van Eden, Claire J. P. Boog, Els J. M. Hogervorst, Marca H. M. Wauben, Ruurd van der Zee, Jan D. A. van Embden
Since mycobacterial species are notoriously difficult to grow in vitro, molecular cloning of their antigens seemed the solution for the development of diagnostics and vaccines. Thole et al. have been involved in cloning antigens of Mycobacterium bovis BCG, the strain used for tuberculosis vaccines, in Escherichia coli K12. A total of six different antigens were expressed as 30, 65, 70, 90, 95, and more than 100 kD molecular weights (11). From serology in mycobacterial diseases one antigen, the 65 kD protein, was already identified as a potent immunogen. By further subcloning of the latter coding gene an overproducing strain was obtained. From this strain the 65 kD could easily be purified and subsequently further characterized (12). By testing this particular protein in proliferation assays, the protein was found to stimulate A2, A2b, and A2c to an extent that exceeded even the stimulation obtained with the original selecting antigen, whole M. tuberculosis (13). All other mycobacterial recombinant proteins available to us at that time were found negative. Sequencing of the M. bovis BCG 65 kD protein coding gene revealed that it was composed of 540 amino acids and that it was identical with the M. tuberculosis 65 kD protein. Furthermore, the protein turned out to have a homology of more than 95% with its Mycobacterium leprae-derived counterpart. Thus, the antigen found responsible for triggering our arthritis T cells turned out to be a protein molecule conserved between various mycobacterial species. Furthermore, a panel of both polyclonal and monoclonal antibodies with specificity for this 65 kD mycobacterial protein was found to recognize, in Western blots, antigens of similar molecular weights in many other bacterial species. These species included arthritis-associated species, such as streptococci, Klebsiella, Shigella, Yersinia, gonococci, and Campylobacter. From these observations it was concluded that the 65 kD protein was a member of a family of proteins called “common antigen” (14). Further analysis revealed extensive sequence homology with the GroEL protein of E. coli, which is a so-called heat-shock or stress protein (15,16). The latter GroEL protein is a major essential protein present in virtually all bacterial cells. The expression of the GroEL gene(s) is upregulated under conditions that are stressful for the organism. The GroEL was also found to be a major protein present in mitochondria of eukaryotic cells, and also in plant chloroplasts. The reason for the evolutionary conservation of these molecules is probably related to their function as so-called molecular chaperones, which are essential to cell integrity, being involved in the assembly and possibly intracellular translocation of other multisubunit proteins.
Analysis of the Escherichia coli extracellular vesicle proteome identifies markers of purity and culture conditions
Published in Journal of Extracellular Vesicles, 2019
Jiwon Hong, Priscila Dauros-Singorenko, Alana Whitcombe, Leo Payne, Cherie Blenkiron, Anthony Phillips, Simon Swift
The 60 kDa chaperonin GroEL, which is required for proper protein folding, was the only protein over-represented in the crude input EVs from both UPEC and Nissle cultured in R. GroEL is a major heat shock protein of E. coli, but abundant and essential even when the cells are not stressed [44]. GroEL could be in crude input EVs because it is complexed with misfolded or damaged proteins that are being excreted. Or it could be a contaminant of preparations from extra-vesicle aggregates or from lysed cells that stick to the outside of EV. If it is confirmed to be a contaminating waste protein, it could be used as a technical marker for purification. However, this protein has been previously detected in many EV studies on Gram-negative bacteria [28] including E. coli strains such as non-pathogenic DH5α [40], Nissle [41], MG1655 [45]; enterohemorrhagic O157 [46] and extraintestinal pathogenic O25b:H4-ST131 [47]. Most of these studies used DGC for purification, although none compared its protein amount before and after the DGC. Further, a recently released commercial kit for bacterial EV isolation (System Biosciences, Cat#EXOBAC100A-1) uses GroEL as an EV marker. Further tests to validate the presence of GroEL as a true-EV associated protein (e.g. using antibody-mediated microscopy techniques to localize GroEL in crude and purified EVs) now seem warranted.
The antigenicity performance of divalent recombinant B. melitensis vaccines versus univalent ones
Published in Alexandria Journal of Medicine, 2019
Tooba Abbassi-Daloii, Soheil Yousefi, Mojtaba Tahmoorespur, Mohammad Hadi Sekhavati
These results are in agreement with some other reports that have shown efficient immune responses against Brucella associated with high levels of Th1 cytokines and IgG2a [7,13]. Luo et al. [20] and Luo et al. [23] showed divalent genetic vaccines elicit stronger cellular immune response and better protection against Brucella abortus than univalent vaccines. Also, Tadepalli et al. [1] studied immunogenicity efficacy of rOmp19, rP39, and rOmp19 + rP39 injections. They found rOmp19 + rP39 immunized mice induced significantly higher proliferative response with considerable cytokines expression, higher IgG2a antibody titer than univalent injected groups, as well. In another study, the simultaneous injection of HSP60 and chimeric BLS-OMP25 antigen showed better immunity than univalent injections of each recombinant protein [20]. However, Abbassi-Daloii et al. [24] found that the simultaneous injection of GroEL with OMP31 and OMP25 did not improve immune responses rather than individual injections of OMP31 and OMP25.
Hydrogen deuterium exchange mass spectrometry applied to chaperones and chaperone-assisted protein folding
Published in Expert Review of Proteomics, 2019
Florian Georgescauld, Thomas E. Wales, John R. Engen
Chaperonins constitute an essential class of chaperones formed by two identical rings of about 0.5 MDa each, stacked back to back. Two groups of chaperonins exist: group I chaperonins present in bacteria (GroEL/GroES), mitochondria (Hsp60/Hsp10) and chloroplasts (Cpn60/Cpn10), and group II chaperonins present in eukaryotes (TriC also called CCT) and archaea (thermosome). GroEL/GroES, the most studied chaperonin, is the only essential molecular chaperone in E. coli and interacts with 250 different cytosolic proteins [16]. About 80 of the 250 proteins are ‘strictly dependent’ substrates (or class III substrates) because at 37°C they are unable to acquire their native structure without help from GroEL/GroES and aggregate rapidly. Natural substrates of GroEL/GroES are frequently proteins that include a TIM-barrel domain, with half of the chaperone substrates being of this variety. GroEL consists of a double ring of seven identical subunits forming a central cavity that can hold proteins between 20 and 60 kDa in size. In the presence of ATP and GroES, one substrate protein at a time will be encapsulated for about 10 s in the ‘nano-compartment’ formed by GroEL/GroES, so that folding can occur. Determining how GroEL/GroES actively promotes folding of the encapsulated substrate protein is a key question in the protein folding field. The eukaryotic cognate of GroEL is the chaperonin TRiC, composed of two rings of eight different paralogous subunits (reviewed in [17]). No analogous GroES lid covers the rings in TRiC, but a built-in lid is formed from apical protrusions of each subunit. Substrates of prokaryotic and eukaryotic chaperonins are different, suggesting different functional mechanisms.