Refolding and characterization of methionine adenosyltransferase from Euglena gracilis

Garrido, Federico; Estrela, Sylvie; Alves, Cláudia; Sánchez-Pérez, Gabino F.; Sillero, Antonio; Pajares, Marı́a A.

doi:10.1016/j.pep.2011.05.004

Cited by 14 publications

(8 citation statements)

References 42 publications

(72 reference statements)

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“…Refolding yield of active oligomeric proteins from IBs is even lower (Scrofani et al, 2000; Karumuri et al, 2007; Garrido et al, 2011). Formation of active monomer and its association is a prerequisite for refolding into fully active oligomeric proteins.…”

Section: Introductionmentioning

confidence: 99%

Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

et al. 2014

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A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form.

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Section: Introductionmentioning

confidence: 99%

Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

et al. 2014

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“…4B; Fisher's exact test, p < 1.00×10 − 3 ), implying that A. veronii could directly manipulate host metabolic process, component organization and homeostasis to achieve ClpP and Prx are also found in almost every organism. The three proteins perform functions by forming homo-oligomer, tetradecamer complex and homo-dimer, respectively [29][30][31]. In our data, A. veronii MetK, ClpP and Prx2 interacted with O. niloticus MetK, ClpP and Prx1, respectively.…”

Section: Virulence Factors May Manipulate Host Biological Processes By Mimicking and Competitively Binding Host Proteinsmentioning

confidence: 55%

Network-Based Analysis of Virulence Factors for Uncovering Aeromonas Veronii Pathogenesis

Tang

et al. 2021

Preprint

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Background: Aeromonas veronii is a pathogen that causes serious harm to aquaculture. Virulence factors are its pathogenic basis, which could promote pathogens to colonize the host, evade host defense and so on. But because experimental verification of virulence factors is time-consuming and laborious, the number of known virulence factors is limited. In this past, most studies only focused on single virulence factor, resulting the biased interpretation for pathogenesis.Results: In this study, a protein-protein interaction (PPI) network at genome-wide scale for A. veronii was first constructed. Then, virulence factors were predicted and mapped on the network. Topological characteristics of the virulence factors were analyzed. The results showed that the virulence factors had higher degree and betweenness centrality than the other proteins in the network. In particular, the virulence factors tended to interact with each other and were enriched in two network modules. One of the modules mainly consisted of histidine kinases, response regulators, diguanylate cyclases and phosphodiesterases, which played important roles in two-component regulatory systems and the synthesis and degradation of cyclic-diGMP. Furthermore, an interspecies PPI network between A. veronii and its host Oreochromis niloticus was also constructed. The structures and interacting sites of the virulence factors and host proteins were added to the interspecies PPI network. By analyzing the interspecies PPI network, we found that the virulence factors could competitively bind host proteins and some of the interacting sites of the virulence factors were shared by different host proteins. Drugs could be designed to target these sites and further prevent pathogen to interfere with host pathways.Conclusions: Our results indicated that the virulence factors regulated the virulence of A. veronii by involving in signal transduction pathway and manipulate host biological processes by mimicking and competitively binding host proteins. Our results deepened the understanding for pathogenesis and had important theoretical significance for designing targeted antibacterial drugs.

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“…In contrast, Met-Ala-(His) 6 -α2 showed a ∼5 fold reduction in the specific activity to 35.19 nmol/min/mg, thus suggesting an effect of the N-terminal tag on this parameter. Addition of tags has been shown to affect both activity and expression of α1 subunits [10] and of other MATs [38], therefore given the high degree of identity among MAT catalytic subunits a similar behavior can be expected. The reason for a longer N-terminal to decrease MAT specific activity is not clear, but may rely on subtle changes in folding affecting indirectly the active site.…”

Section: Resultsmentioning

confidence: 98%

NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

et al. 2012

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Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

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Refolding and characterization of methionine adenosyltransferase from Euglena gracilis

Abstract: KeywordsMethionine adenosyltransferase, inclusion bodies, refolding, characterization, secondary structure, structural model.

Cited by 14 publications

References 42 publications

Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

Network-Based Analysis of Virulence Factors for Uncovering Aeromonas Veronii Pathogenesis

NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

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