A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.     

The Effects of Organic Solvent on the Ribosyl Transfer Reaction by Thermostable Purine Nucleoside Phosphorylase and Pyrimidine Nucleoside Phosphorylase from Bacillus stearothermophilus JTS 859

The effects of organic solvents on the reaction rate and equilibrium of the ribosyl transfer reaction catalyzed by thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859 were examined at 60°C. The reaction rate in the presence of 10% acetone was 1.6 times higher than that of the control. Acetone was the best organic solvent among those tested for accelerating the reaction rate without denaturing the enzymes. On the other hand, the reaction rate in the presence of 5% ethyl acetate was 1.5 times higher than that of the control. However the enzymes were denatured completely after 1 h incubation. Consequently, the acceleration was not attributed to the stabilization of the enzymes. The equilibrium constants of the reaction were not influenced by the presence of acetone, methyl or ethyl alcohols.     

Enzymes of ammonia assimilation in legume nodules: A comparison between ureide- and amide-transporting plants

Levels of amide and ureide biogenic enzymes were compared in the plant cytosol fractions of root nodules from soybean ( Glycine max L. Merr., cv. Williams), pintobean ( Phaseolus vulgaris L. cv. Pinto) and Lupin ( Lupinus angustifolius L. cv. Frost). Enzymes of purine oxidation were found to be present in significant quantities only in ureide-transporting pintobean and soybean nodules. The levels of these enzymes were low in lupin, but this amide-exporter had significantly higher levels of asparagine synthetase. Enzymes of de novo purine biosynthesis and glycine biosynthesis were present at higher levels in pintobean and soybean, consistent with a role for de novo purine biosynthesis in ureide biogenesis. The low levels of these enzymes in lupin are consistent with a role in general purine and amino acid metabolism in these nodules, not directly related to the synthesis of transport compounds for fixed atmospheric nitrogen. Amino acid concentrations in soybean, pintobean and lupin nodules reflected the metabolic differences between amide and ureide plants. The comparative data presented are consistent with a pathway of ureide biogenesis using glutamine, glutamate and aspartate synthesized via reactions catalyzed by glutamine synthetase, glutamate synthase and aspartate aminotransferase in the de novo synthesis of purines followed by oxidation of these purines to produce the ureides allantoin and allantoic acid.     

Regioselective Enzymatic Hydrolysis of 3',5'-DI-O-Acetylthymidine and Observation of a Surface Effect Due to Polystyrene

The selective enzymatic hydrolysis of 3',5'-di-O-acetylthyidine (1) was studied. The lipases from porcine pancreas and Aspergillus niger, and pig liver esterase, all catalysed selective hydrolysis of the 5'O-acetyl group, but the lipase from Candida cylindracea catalysed selective hydrolysis of the 3'-O-acetyl group. Highest selectivity, leading to essentially pure 3'-O-acetylthymidine, was achieved using porcine pancreatic lipase in dilute solution at pH 7.5. Provision of an artificial interface in the form of polystyrene beads led to a significant increase in the rate of hydrolysis, accompanied by a marked fall in selectivity. Other changes in the hydrolysis conditions, such as raising the concentration of substrate or adding cosolvent, also led to a fall in selectivity.     

Synthesis of 2-(3′-Azido- and 3′-Amino-3′-deoxy-β-D-ribofuranosyl)thiazole-4-carboxamide

Abstract

In view of biological activities of tiazofurin and azido or aminosugar nucleosides, novel azido- and amino-substituted tiazofurin derivatives (1 and 2) were efficiently synthesized starting from 1,2;5,6-di-O-isopropylidene-D-glucose.     

Synthesis of Carbocyclic Analogues of MECA and NECA 1,2-Disubstituted as Potential Adenosine Receptor Agonists

Abstract

A new class of 1,2-disubstituted carbocyclic nucleosides of MECA and NECA analogues was synthesized in gool yield starting from (±) 6-azabicyclo[3.2.0]heptan-7-one.     

Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine

Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential. We have employed resonance Raman spectroscopy at an excitation wavelength of 260 nm, to decipher solution structure of 9-deazaguanine (9DAG) and 9-deazahypoxanthine (9DAH). These are analogues of guanine and hypoxanthine, respectively, and have been exploited to study static complexes of nucleobase binding enzymes. Such enzymes are known to perturb pK_a of their ligands, and thus, we also determined solution structures of these analogues at two, acidic and alkaline, pH. Structure of each possible protonation state and tautomer was computed using density functional theoretical calculations. Species at various pHs were identified based on isotopic shifts in experimental wavenumbers and by comparing these shifts with corresponding computed isotopic shifts. Our results show that at physiological pH, N1 of pyrimidine ring in 9DAG and 9DAH bears a proton. At lower pH, N3 is place of protonation, and at higher pH, deprotonation occurs at N1 position. The proton at N7 of purine ring remains intact even at pH 12.5. We have further compared these results with naturally occurring nucleotides. Our results identify key vibrational modes which can report on hydrogen bonding interactions, protonation and deprotonation in purine rings upon binding to the active site of enzymes.     

The Reaction of Sulfite Radical Anion with Nucleic Acid Components

The sulfite radical anion (SO₃^?) is the first intermediate in the autoxidation of sulfite to sulfate. Using competition kinetics, its reactivities with the nucleic acid bases and the corresponding nucleosides were investigated. The second order rate constants were found to be rather low, k < 1 × 10⁶dm³mol^?1s^?1 at pH 7. As a competitor, the carotenoid crocin was used, which was found to be bleached very efficiently by SO₃^? (k = 1.0 × 10⁹dm³mol^?1S^?1).     

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.     

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.