1,720,974 research outputs found
A HIGHLY STABLE, PROTEASE-RESISTANT E. COLI ASPARAGINASE
No full textAn invention which can help the treatment of Acute Lymphoblastic Leukaemia
Helicobacter pylori L-asparaginase: A Novel Bacterial Antigen that May Contribute to Infection Detection
No full textHelicobacter pylori is responsible for gastric inflammation and for an increased risk of cancer development in humans. Several bacterial antigens contribute to stimulate the immune system, but their relative role has not yet been defined. H. pylori (strain CCUG) type II L-asparaginase (L-ASNase) induces an immune response in mice. To verify if an immune response could also be detected in humans, sera positive (n=11) or negative (n=11), respectively, to H. pylori according to a commercial test were assayed for their reactivity towards purified H. pylori L-ASNase. Among positive samples, 8/11 (72%) were positive to L-ASNase. We conclude that H. pylori L-ASNase is immunogenic in humans and contributes to the generation of the antibody response induced by the bacterium
Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies
No full textHeterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007)
Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.
No full textSingle domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes
Structural Aspects of E. coli Type II Asparaginase in Complex with Its Secondary Product L-Glutamate
No full textBacterial L-asparaginases are amidohydrolases (EC 3.5.1.1) capable of deaminating L-asparagine and, with reduced efficiency, L-glutamine. Interest in the study of L-asparaginases is driven by their use as biodrugs for the treatment of acute lymphoblastic leukemia. Here, we report for the first time the description of the molecular structure of type II asparaginase from Escherichia coli in complex with its secondary product, L-glutamate. To obtain high-quality crystals, we took advantage of the N24S variant, which has structural and functional features similar to the wild-type enzyme, but improved stability, and which yields more ordered crystals. Analysis of the structure of the N24S-L–glutamate complex (N24S–GLU) and comparison with its apo and L-aspartate-bound form confirmed that the enzyme-reduced catalytic efficiency in the presence of L-glutamine is due to L-glutamine misfitting into the enzyme-binding pocket, which causes a local change in the catalytic center geometry. Moreover, a tight interaction between the two protomers that form the enzyme active site limits the capability of L-glutamine to fit into (and to exit from) the binding pocket of E. coli L-asparaginase, explaining why the enzyme has lower glutaminolytic activity compared to other enzymes of the same family, in particular the Erwinia chrysanthemi one
High Resolution Structure of Human Apolipoprotein (a) Kringle IV Type 2: Beyond the Lysine Binding Site
No full textLipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apolipoprotein (a) [apo(a)]. The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analogue epsilon-aminocaproic acid (EACA). Interestingly, KIV-2 presents a Lysine Binding Site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in Surface Plasmon Resonance (SPR) experiments, and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS" (pLBS). Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a) related diseases and to facilitate the design of specific therapeutic drugs. (226 words)
Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase
Full text linkBacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.
No full textBacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy
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