Browsing by Subject "PURIFICATION"

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  • Beyer, Hannes M.; Iwai, Hideo (2019)
    Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein-splicing activity are associated with bottlenecks such as the selection of pseudo-positive clones arising from off-pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein-splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis- and trans-splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed-evolution approaches or the natural intein sequence space.
  • Kuryk, Lukasz; Moller, Anne-Sophie W.; Vuolanto, Antti; Pesonen, Sari; Garofalo, Mariangela; Cerullo, Vincenzo; Jaderberg, Magnus (2019)
    Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm(2) (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 +/- 100 and 14,559 +/- 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 x 10(9) +/- 0.2 and 1.75 x 10(9) +/- 0.08 infectious particles of ONCOS-401 per cm(2) of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses.
  • Pulkkinen, Marjo; Coda, Rossana; Lampi, Anna-Maija; Varis, Jutta; Katina, Kati; Piironen, Vieno (2019)
    Vicine and convicine may be removed from faba bean by hydrolysis to the corresponding aglycones, divicine and isouramil. For total elimination of their toxicity, further degradation of the aglycones should be shown. The aim of the study was to investigate hydrolysis of vicine and convicine using the enzymatic activity in faba bean in flour suspensions and selected lactic acid bacteria used as starters for faba bean fermentation. In addition, the effect of acidity on the stability of vicine and convicine was investigated. Sourdoughs were used in a baking process to obtain breads as final products. Vicine, convicine, and their aglycones were analyzed using reversed phase high pressure liquid chromatography with UV detection (RP-HPLC-UV). Incubation of the suspensions showed rather small vicine and convicine losses. Acidity itself did not cause losses under the conditions studied, apart from that of convicine at low pH. In sourdough fermentation with strains of Lactobacillus plantarum and Pediococcus pentosaceus, losses of vicine and convicine were dependent on the fermentation temperature and the β-glucosidase activity of the starter. Compared to fermentation at 20 °C, more intense acidification at 25 °C resulted in decrease of vicine up to 85% and convicine up to 47%. Levels of vicine and convicine in breads were comparable to levels in sourdoughs. Furthermore, the aglycones were not detected from breads.
  • Cononi Linares, Nancy; Dilokpimol, Adiphol; Stålbrand, Henrik; Mäkelä, Miia; de Vries, Ronald (2020)
    alpha-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular alpha-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All alpha-galactosidases exhibited high affinity to pNP alpha Gal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining alpha-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other alpha-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal alpha-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses.
  • Syed, Shahan; Nissilä, Eija; Ruhanen, Hanna; Fudo, Satoshi; Gaytan, Meztlli O.; Sihvo, Sanna P.; Lorey, Martina B.; Metso, Jari; Oorni, Katariina; King, Samantha J.; Oommen, Oommen P.; Jauhiainen, Matti; Meri, Seppo; Käkelä, Reijo; Haapasalo, Karita (2021)
    High-density lipoproteins (HDLs) are a group of different subpopulations of sialylated particles that have an essential role in the reverse cholesterol transport (RCT) pathway. Importantly, changes in the protein and lipid composition of HDLsmay lead to the formation of particles with reduced atheroprotective properties. Here, we show that Streptococcus pneumoniae pneumolysin (PLY) and neuraminidase A (NanA) impair HDL function by causing chemical and structural modifications of HDLs. The proteomic, lipidomic, cellular, and biochemical analysis revealed that PLY and NanA induce significant changes in sialic acid, protein, and lipid compositions of HDL. The modified HDL particles have reduced cholesterol acceptor potential from activated macrophages, elevated levels of malondialdehyde adducts, and show significantly increased complement activating capacity. These results suggest that accumulation of these modified HDL particles in the arterial intima may present a trigger for complement activation, inflammatory response, and thereby promote atherogenic disease progression.
  • Oeemig, Jesper S.; Beyer, Hannes M.; Aranko, A. Sesilja; Mutanen, Justus; Iwai, Hideo (2020)
    Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the -1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.
  • Hietala, Ville; Horsma-Heikkinen, Jenni; Carron, Annelie; Skurnik, Mikael; Kiljunen, Saija (2019)
    The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiMiHyAci03, and Staphylococcus phage vB_SauMiRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_HoEco02 lysate from 3.5 x 10(4) Endotoxin Units (EU)/10(9) plaque forming units (PFU) to 0.09 EU/10 9 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/10(9) PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/10(9) PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.
  • Patrikainen, Maarit; Pan, Peiwen; Kulesskaya, Natalia; Voikar, Vootele; Parkkila, Seppo (2014)
  • Rice, S.; Algora, A.; Tain, J. L.; Valencia, E.; Agramunt, J.; Rubio, B.; Gelletly, W.; Regan, P. H.; Zakari-Issoufou, A. -A.; Fallot, M.; Porta, A.; Rissanen, J.; Eronen, T.; Äystö, J.; Batist, L.; Bowry, M.; Bui, V. M.; Caballero-Folch, R.; Cano-Ou, D.; Elomaa, V. -V.; Estevez, E.; Farrelly, G. F.; Garcia, A. R.; Gomez-Hornillos, B.; Gorlychev, V.; Hakala, J.; Jordan, M. D.; Jokinen, A.; Kolhinen, V. S.; Kondev, F. G.; Martinez, T.; Mason, P.; Mendoza, E.; Moore, I.; Penttilä, H.; Podolyak, Zs.; Reponen, M.; Sonnenschein, V.; Sonzogni, A. A.; Sarriguren, P. (2017)
    The beta decays of Br-86 and Rb-91 have been studied using the total absorption spectroscopy technique. The radioactive nuclei were produced at the Ion Guide Isotope Separator On-Line facility in Jyvaskyla and further purified using the JYFLTRAP. Br-86 and Rb-91 are considered to be major contributors to the decay heat in reactors. In addition, Rb-91 was used as a normalization point in direct measurements of mean gamma energies released in the beta decay of fission products by Rudstam et al. assuming that this decaywas well known from high-resolution measurements. Our results show that both decays were suffering from the Pandemonium effect and that the results of Rudstam et al. should be renormalized. The relative impact of the studied decays in the prediction of the decay heat and antineutrino spectrum from reactors has been evaluated.
  • Kasurinen, Aaro; Laitinen, Alli; Kokkola, Arto; Stenman, Ulf-Håkan; Böckelman, Camilla; Haglund, Caj (2020)
    Introduction: Tumor-associated trypsin inhibitor (TATI) limits serine proteases, promotes carcinogenesis in several cancers and functions as an acute-phase reactant. Tumor-associated trypsin-2 (TAT-2), a proteolytic target enzyme for TATI, can enhance invasion by promoting extracellular matrix degradation. Here, we aimed to study serum TATI and TAT-2 levels, including the TAT-2/TATI ratio, as prognostic and diagnostic biomarkers in gastric cancer. We compared the results with the plasma level of C-reactive protein (CRP). Material and Methods: We selected 240 individuals operated on for gastric adenocarcinoma at the Helsinki University Hospital, Finland, between 2000 and 2009. We determined the preoperative serum TAT-2, TATI and plasma CRP levels using time-resolved immunofluorometric assays using monoclonal antibodies. Results: The medium serum TAT-2 level was higher among gastric cancer patients [8.68 ng/ml; interquartile range (IQR) 5.93-13.2] than among benign controls (median 5.41 ng/ml; IQR 4.12-11.8; p = .005). Five-year survival among patients with a high serum TAT-2 was 22.9% [95% confidence interval (CI) 11.7-34.1], compared to 52.2% (95% CI 44.6-59.8; p <.001) among those with a low level. The five-year survival among patients with a high serum TATI was 30.6% (95% CI 20.4-40.8), compared to 52.9% (95% CI 44.7-61.1; p <.001) among those with a low level. The serum TATI level remained significant in the multivariable survival analysis (hazard ratio 2.01; 95% CI 1.32-3.07). An elevated plasma CRP level associated with a high serum TATI level (p = .037). Conclusions: This study shows for the first time that a high serum TAT-2 may function as a prognostic biomarker in gastric cancer and that TAT-2 levels may be elevated compared to controls. Additionally, we show that the prognosis is worse among gastric cancer patients with a high serum TATI. These biomarkers serve as prognostic factors particularly among patients with a metastatic or a locally advanced disease.