1,721,133 research outputs found
Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197
Full text linkDuring 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19
STRUCTURAL CHARACTERISATION OF M-PARTLY FOLDED INTERMEDIATES OF WILD-TYPE AND P138A ECASPAT
No full text
STRUCTURAL CHARACTERISATION OF M-PARTLY FOLDED INTERMEDIATES OF WILD-TYPE AND P138A ECASPAT
No full textSTRUCTURAL CHARACTERISATION OF M-PARTLY FOLDED INTERMEDIATES OF WILD-TYPE AND P138A ECASPAT
No full textSubstrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from Mycobacterium tuberculosis
Full text linkMycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis-Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site
Structural characterization of the M* partly folded intermediate of wild-type and P138A EcAspAT
Full text linkA combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M*partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M*intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N2 forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposabl
Stabilization of the Escherichia coli DNA polymerase III epsilon subunit by the theta subunit favors in vivo assembly of the Pol III catalytic core
No full textEscherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: alpha (polymerase). epsilon (3'-5' exonuclease), and theta. Genetic experiments suggested that theta subunit stabilizes the intrinsically labile epsilon subunit and, furthermore, that theta might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a Delta holE strain (lacking theta subunit) displays reduced amounts of free epsilon. We also demonstrate the existence of a dimer of epsilon, which may be involved in the stabilization of the protein. Second, theta, when overexpressed, dissociates the epsilon dimer and significantly increases the amount of Pol III core. The stability oft epsilon also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of Delta dnaK strains leads to rapid depletion of E, and (ii) overproduction of theta overcomes both the depletion of epsilon, and the temperature sensitivity of the strain. Overall, our data suggest that epsilon is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the theta subunit through its prevention oft epsilon degradation
The assembly of Escherichia coli DNA polymerase III catalytic core
No full textThis study is mainly focused on the assembly of the DNA polymerase III catalytic core of Escherichia coli. This enzyme is responsible for the chromosome replication and it is constituted of multiple subunits. In particular, the polymerase and the exonuclease (proofreading) activities are located into two different subunits: (coded by dnaE gene) and , (coded by dnaQ). The catalytic core is composed also of the subunit with an accessory role and a dispensable nature. On the contrary, the dnaE and dnaQ are essential genes and their interaction is crucial for the assembly of a DNA polymerase III active enzyme. Several data are available on the properties of the DNA polymerase III in vitro, while little information concerns the assembly in vivo of this enzyme. The present work investigated the interaction of alpha and epsilon subunits by using different truncated forms produced in vivo, in order to identify which residues of epsilon (DnaQ) are essential for binding to alpha. Moreover, the stability of the mutant proteins was analyzed and we found that the C-terminal region of epsilon is labile and affected by proteolysis. In addition, different factors (proteases and chaperones) potentially involved in the regulation of epsilon stability have been studied
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