IU Indianapolis ScholarWorks :: Browsing by Subject "Recombinant proteins"

Browsing by Subject "Recombinant proteins"

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A Dose-Escalation Study of Recombinant Human Interleukin-18 Using Two Different Schedules of Administration in Patients with Cancer
(American Association for Cancer Research, 2008-06) Robertson, Michael J.; Kirkwood, John M.; Logan, Theodore F.; Koch, Kevin M.; Kathman, Steven; Kirby, Lyndon C.; Bell, William N.; Thurmond, Linda M.; Weisenbach, Jill; Dar, Mohammed M.; Medicine, School of Medicine
Purpose: Interleukin-18 (IL-18) is an immunostimulatory cytokine with antitumor activity in preclinical models. A phase I study of recombinant human IL-18 (rhIL-18) was done to determine the toxicity, pharmacokinetics, and biological activities of rhIL-18 administered at different doses in two different schedules to patients with advanced cancer. Experimental design: Cohorts of three to four patients were given escalating doses of rhIL-18 as a 2-h i.v. infusion either on 5 consecutive days repeated every 28 days (group A) or once a week (group B) for up to 6 months. Toxicities were graded using standard criteria. Blood samples were obtained for safety, pharmacokinetic, and pharmacodynamic measurements. Results: Nineteen patients (10 melanoma and 9 renal cell cancer) were given rhIL-18 in doses of 100, 500, or 1,000 microg/kg (group A) or 100, 1,000, or 2,000 microg/kg (group B). Common side effects included chills, fever, headache, fatigue, and nausea. Common laboratory abnormalities included transient, asymptomatic grade 1 to 3 lymphopenia, grade 1 to 4 hyperglycemia, grade 1 to 2 anemia, neutropenia, hypoalbuminemia, liver enzyme elevations, and serum creatinine elevations. No dose-limiting toxicities were observed. Biological effects of rhIL-18 included transient lymphopenia and increased expression of activation antigens on lymphocytes. Increases in serum concentrations of IFN-gamma, granulocyte macrophage colony-stimulating factor, and IL-18-binding protein were observed following dosing. Conclusions: rhIL-18 can be given in biologically active doses by either weekly infusions or daily infusions for 5 days repeated every 28 days to patients with advanced cancer. Toxicity was generally mild to moderate, and a maximum tolerated dose of rhIL-18 by either schedule was not determined.
Formation of Hexacoordinate Mn(III) in Bacillus subtilis Oxalate Decarboxylase Requires Catalytic Turnover
(American Chemical Society, 2016-01-26) Zhu, Wen; Wilcoxen, Jarett; Britt, R. David; Richards, Nigel G.J.; Chemistry and Chemical Biology, School of Science
Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C-C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).
IMP Dehydrogenase from the Protozoan Parasite Toxoplasma gondii
(American Society for Microbiology, 2005) Sullivan, William J., Jr.; Dixon, Stacy E.; Li, Catherine; Striepen, Boris; Queener, Sherry F.; Pharmacology and Toxicology, School of Medicine
The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.
MYST family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinase-mediated DNA damage response in Toxoplasma gondii
(Elsevier, 2010) Vonlaufen, Nathalie; Naguleswaran, Arunasalam; Coppens, Isabelle; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
The MYST family of lysine acetyltransferases (KATs) function in a wide variety of cellular operations, including gene regulation and the DNA damage response. Here we report the characterization of the second MYST family KAT in the protozoan parasite Toxoplasma gondii (TgMYST-B). Toxoplasma causes birth defects and is an opportunistic pathogen in the immunocompromised, the latter due to its ability to convert into a latent cyst (bradyzoite). We demonstrate that TgMYST-B can gain access to the parasite nucleus and acetylate histones. Overexpression of recombinant, tagged TgMYST-B reduces growth rate in vitro and confers protection from a DNA-alkylating agent. Expression of mutant TgMYST-B produced no growth defect and failed to protect against DNA damage. We demonstrate that cells overexpressing TgMYST-B have increased levels of ataxia telangiectasia mutated (ATM) kinase and phosphorylated H2AX and that TgMYST-B localizes to the ATM kinase gene. Pharmacological inhibitors of ATM kinase or KATs reverse the slow growth phenotype seen in parasites overexpressing TgMYST-B. These studies are the first to show that a MYST KAT contributes to ATM kinase gene expression, further illuminating the mechanism of how ATM kinase is up-regulated to respond to DNA damage.
Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii
(Elsevier, 2004) Chaudhary, Kshitiz; Darling, John A.; Fohl, Leah M.; Sullivan, William J., Jr.; Donald, Robert G. K.; Pfefferkorn, Elmer R.; Ullman, Buddy; Roos, David S.; Pharmacology and Toxicology, School of Medicine
We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.
Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase
(Elsevier, 1999) Darling, John A.; Sullivan, William J., Jr.; Carter, Darrick; Ullman, Buddy; Roos, David S.
Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.
Specific Lowering of Asymmetric Dimethylarginine by Pharmacological Dimethylarginine Dimethylaminohydrolase Improves Endothelial Function, Reduces Blood Pressure and Ischemia-Reperfusion Injury
(American Society for Pharmacology and Experimental Therapeutics, 2021) Lee, Young; Mehrotra, Purvi; Basile, David; Ullah, Mahbub; Singh, Arshnoor; Skill, Nicholas; Younes, Subhi Talal; Sasser, Jennifer; Shekhar, Anantha; Singh, Jaipal; Cellular and Integrative Physiology, School of Medicine
Multiple clinical and preclinical studies have demonstrated that plasma levels of asymmetric dimethylarginine (ADMA) are strongly associated with hypertension, diabetes, and cardiovascular and renal disease. Genetic studies in rodents have provided evidence that ADMA metabolizing dimethylarginine dimethylaminohydrolase (DDAH)-1 plays a role in hypertension and cardiovascular disease. However, it remains to be established whether ADMA is a bystander, biomarker, or sufficient contributor to the pathogenesis of hypertension and cardiovascular and renal disease. The goal of the present investigation was to develop a pharmacological molecule to specifically lower ADMA and determine the physiologic consequences of ADMA lowering in animal models. Further, we sought to determine whether ADMA lowering will produce therapeutic benefits in vascular disease in which high ADMA levels are produced. A novel long-acting recombinant DDAH (M-DDAH) was produced by post-translational modification, which effectively lowered ADMA in vitro and in vivo. Treatment with M-DDAH improved endothelial function as measured by increase in cGMP and in vitro angiogenesis. In a rat model of hypertension, M-DDAH significantly reduced blood pressure (vehicle: 187 ± 19 mm Hg vs. M-DDAH: 157 ± 23 mm Hg; P < 0.05). Similarly, in a rat model of ischemia-reperfusion injury, M-DDAH significantly improved renal function as measured by reduction in serum creatinine (vehicle: 3.14 ± 0.74 mg/dl vs. M-DDAH: 1.1 ± 0.75 mg/dl; P < 0.01), inflammation, and injured tubules (vehicle: 73.1 ± 11.1% vs. M-DDAH: 22.1 ± 18.4%; P < 0.001). These pharmacological studies have provided direct evidence for a pathologic role of ADMA and the therapeutic benefits of ADMA lowering in preclinical models of endothelial dysfunction, hypertension, and ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: High levels of ADMA occur in patients with cardiovascular and renal disease. A novel modified dimethylarginine dimethylaminohydrolase by PEGylation effectively lowers ADMA, improves endothelial function, reduces blood pressure and protects from ischemia-reperfusion renal injury.
Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis
(Elsevier, 1997) Roos, David S.; Sullivan, William J., Jr.; Striepen, Boris; Bohne, Wolfgang; Donald, Robert G. K.
Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase-thymidylate synthase (DHFR-TS) locus of Toxoplasma gondii integrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may-but need not-include mutant DHFR-TS alleles that confer pyrimethamine resistance to transgenic parasites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast, Drosophila, etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form of T. gondii and for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to "trap" genes that are activated (or inactivated) under various conditions of interest.
The adenosine transporter of Toxoplasma gondii: Identification by insertional mutagenesis, cloning, and recombinant expression
(Elsevier, 1999) Chiang, Chi-Wu; Carter, Nicola; Sullivan, William J., Jr.; Donald, Robert G. K.; Roos, David S.; Naguib, Fardos N. M.; el Kouni, Mahmoud H.; Ullman, Buddy; Wilson, Craig M.
Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A-resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 microM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.
Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system
(Public Library of Science, 2018-04-03) Imasaki, Tsuyoshi; Wenzel, Sabine; Yamada, Kentaro; Bryant, Megan L.; Takagi, Yuichiro; Biochemistry and Molecular Biology, School of Medicine
The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

Browsing by Subject "Recombinant proteins"

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