1,354,180 research outputs found
Alejandro Hochkoeppler, Alessandro Scalzo: Metodo per l'estrazione di proteine ricombinanti
No full textL'invenzione riguarda una metodologia, applicabile a livello industriale, per l'estrazione di proteine ricombinanti prodotte mediante Escherichia coli. Tale metodologia permette di isolare rapidamente ed in forma solubile proteine ed enzimi sovraespressi nel summenzionato microorganismo
The catalytic action of human D-lactate dehydrogenase is severely inhibited by oxalate and is impaired by mutations triggering D-lactate acidosis
No full textD-lactate dehydrogenases are known to be expressed by prokaryotes and by eukaryotic invertebrates, and over the years the functional and structural features of some bacterial representatives of this enzyme ensemble have been investigated quite in detail. Remarkably, a human gene coding for a putative D-lactate dehydrogenase (DLDH) was identified and characterized, disclosing the occurrence of alternative splicing of its primary transcript. This translates into the expression of two human DLDH (hDLDH) isoforms, the molecular mass of which is expected to differ by 2.7 kDa. However, no information on these two hDLDH isoforms is available at the protein level. Here we report on the catalytic action of these enzymes, along with a first analysis of their structural features. In particular, we show that hDLDH is strictly stereospecific, with the larger isoform (hDLDH-1) featuring higher activity at the expense of D-lactate when compared to its smaller counterpart (hDLDH-2). Furthermore, we found that hDLDH is strongly inhibited by oxalate, as indicated by a Ki equal to 1.2 mu M for this dicarboxylic acid. Structurally speaking, hDLDH-1 and hDLDH-2 were determined, by means of gel filtration and dynamic light scattering experiments, to be a hexamer and a tetramer, respectively. Moreover, in agreement with previous studies performed with human mitochondria, we identified FAD as the cofactor of hDLDH, and we report here a model of FAD binding by the human D-lactate dehydrogenase. Interestingly, the mutations W323C and T412 M negatively affect the activity of hDLDH, most likely by impairing the enzyme electron-acceptor site
ASFV DNA polymerase extends recessed DNAs with catalytic efficiencies outperforming those exerted on gapped DNA substrates
No full textThe DNA polymerase from african swine fever virus (ASFV Pol X), lacking both 8 kDa and thumb domains, is the smallest enzyme featuring competence in DNA extension. Here we show that ASFV Pol X features poor filling activity of DNA gaps consisting of 15 bases, and exerts a more efficient action at the expense of DNA substrates containing a recessed end of equal length. We also show that shortening the recessed end of DNA substrates decreases the rate of DNA elongation catalysed by ASFV Pol X. Finally, by means of stopped-flow experiments we were able to determine that DNA binding is a step responsible for restraining the efficiency of ASFV Pol X catalytic action
Espressione genica e fisiologia cellulare
No full textIl contributo analizza la regolazione dell'espressione genica descrivendo la costruzione e lo sviluppo del modello dell'operon lac
Amberlite XAD-4 is a convenient tool for removing Triton X-100 and Sarkosyl from protein solutions
No full textAmberlite has been shown to be an appropriate material for the adsorption of organic contaminants from aqueous solutions. In addition, Amberlite XAD-2 has been successfully used, as an alternative to Bio-Beads, to remove Triton X-100 from protein solutions, such as from samples of solubilized membrane proteins. However, Amberlite has not been tested as an adsorbent when a mixture of detergents is necessary to solubilize and refold a target protein. Here the authors show that Amberlite XAD-4 can be appropriately used to aid the purification process of proteins solubilized from inclusion bodies with the ternary detergent system consisting of Sarkosyl, Triton X-100 and CHAPS
Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain
No full textWe report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3’-5’ exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and C M equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs
The four subunits of rabbit skeletal muscle lactate dehydrogenase do not exert their catalytic action additively
No full textOligomeric enzymes containing multiple active sites are usually considered to perform their catalytic action at higher rates when compared with their monomeric counterparts. This implies, in turn, that the activity performed by different holoenzyme subunits features additivity. Nevertheless, the extent of this additivity occurring in holoenzymes is far from being adequately understood. To tackle this point, we used tetrameric rabbit lactate dehydrogenase (rbLDH) as a model system to assay the reduction of pyruvate catalysed by this enzyme at the expense of beta-NADH under pre-steady-state conditions. In particular, we observed the kinetics of reactions triggered by concentrations of beta-NADH equimolar to 1, 2, 3, or all 4 subunits of the rbLDH holoenzyme, in the presence of an excess of pyruvate. Surprisingly, when the concentration of the limiting reactant exceeded that of a single holoenzyme subunit, we observed a sharp slowdown of the enzyme conformational rearrangements associated to the generation and the release of L-lactate. Furthermore, using a model to interpret the complex kinetics observed under the highest concentration of the limiting reactant, we estimated the diversity of the rates describing the action of the different rbLDH subunits
Expression and Purification of Diphtheria Toxin Variant CRM197 in Escherichia coli
No full textThe cross reacting material 197 (CRM197) is a nontoxic variant of the diphtheria toxin (DTx) featuring identical immunological properties and similar ability to bind the heparin-binding epidermal growth factor (HB-EGF). The only difference between CRM197 and DTx is a single amino acid substitution at position 52 (glutamic acid instead of glycine). Due to the absence of toxicity, and to its strong inflammatory-immunological property, the CRM197 protein is currently used in several conjugate vaccines. Diphtheria toxin and other related CRM proteins are generally produced using cultures of Corynebacterium diphteriae infected by specific β-phages carrying the tox gene (wt or mutated, respectively).
Here we propose a new and alternative procedure for the production of CRM197 using Escherichia coli as host strain. This process presents several advantages: a reduced time for the coltivation of bacteria; a simple culture medium; the safety of E. coli bacteria compared to C. diptheriae. To this aim, a synthetic gene coding for CRM197 and optimized for E. coli codon usage, was cloned into a specific vector (pET9a) based on the T7 RNA polymerase system. The over-expression was induced in BL21AI E. coli strain simply by adding arabinose to the culture medium. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilized using urea as denaturant. After the expression and solubilization steps, the refolding and purification conditions were experimentally assayed to define a simple procedure for the production of CRM197 in a pure and active form. In particular, the recombinant protein was purified by two different chromatographic steps (affinity and gel-filtration chromatography) and the purity of the final preparation reached up to 95%
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