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EXPRESSION OF PHENYLALANINE AMMONIA-LYASES IN ESCHERICHIA COLI STRAINS EXPERIMENTAL SECTION Transformation and expression of PAL genes E. coli TOP 10 and Rosetta (DE3) strains were transformed with different plasmids containing PAL genes (see Table 1.) and grown in 50 mL low salt LB broth/medium with 100 μg/mL ampicillin [and 35 μg/mL chlorampehnicol only for Rosetta (DE3) strain] overnight 37°C with shaking (220 rpm) until OD600= ~1.5. From the resulting culture, 800 μL volume was added to 50 mL LB (containing 100 µg/mL ampicillin and 35 μg/mL chlorampehnicol). The resulting cultures were grown at 37°C with vigorous shaking until OD600 = ~0.6. At this OD, the temperature was changed to the induction temperature (in a range of 18 ºC − 37 ºC; 4 ºC steps for screening) and the cells were induced with arabinose (in a range of 0,02-0,2 %; 0.05 % steps for screening). At 4 h after the first induction, the same amount of inductor was added to Rosetta cell cultures. After addition of the inducer, the cultures were further cultivated for 12 h and harvested by centrifugation. All subsequent operations were carried out at 4 ºC. Cell disruption and activity measurement The pellet was resuspended in 5 mL of lysis buffer (150 mM NaCl, 50 mM TRIS pH 8.0, 10 mM BME Protease inhibitor cocktail, 2 mM PMSF, 5 mM BA) and sonicated in ice bath at amplitude 40 % and pulsation 60 % using a Bandelin Sonopuls HD 2070 instrument. The sonication was performed until the viscosity of the suspension was significantly lowered. After centrifugation (30 min at 5000 x g), PAL activity was determined in the crude extract by monitoring the formation of (E)-cinnamate at 290 nm (ε 290 =10 4 L M -1 cm -1 ). The assay contained performed at room temperature by addition of 100 μL of the supernatant from the crude extract to 1000 μL of 0.1 M Tris-HCl, pH 8.8, containing 20 mM L-phenylalanine and recording the absorbance at 290 nm for 10 min. The PAL content of the crude extract, supernatant and pellet were analyzed by SDS-PAGE as well. ACKNOWLEDGMENTS This work was supported by the Hungarian National Office for Research and Technology (NKFP-07-A2 FLOWREAC), by the Hungarian National Science Foundation (OTKA T-048854, K68229 and CK78646) and by Howard Hughes Medical Institutes #55000342. This work is also related to the scientific program of "Development of qualityoriented and harmonized R+D+I strategy and functional model at BME" project (TÁMOP- 4.2.1/B-09/1/KMR-2010-0002), supported by the New Hungary Development Plan. The authors thank Prof. János Rétey (Institute of Organic Chemistry, Karlsruhe Institute of Technology, Germany), Prof. G. E. Schulz and M. Baedeker (University of Freiburg, Germany) for providing us with the Petroselinum crispum PAL expression system,) and also Dr M. Stieger (Hoffmann-La Roche, Basel, Switzerland) for the vector pREP4-GroESL carrying the HSP-60 system. 281
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CHEMIA 4/2010
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L.M. PĂCUREANU, A. BORA, L. CRIŞA
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Studia Universitatis Babes-Bolyai C
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MIRCEA V. DIUDEA theorem in multi-s
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MIRCEA V. DIUDEA 4. M.V. Diudea, Cs
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STUDIA UBB. CHEMIA, LV, 4, 2010 DIA
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DIAMOND D 5 , A NOVEL ALLOTROPE OF
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MONICA L. POP, MIRCEA V. DIUDEA AND
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TO WHAT EXTENT THE NMR “MOBILE PR
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DIAGNOSTIC OF A QSPR MODEL: AQUEOUS
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STUDIA UBB. CHEMIA, LV, 4, 2010 MOD
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MODELING THE BIOLOGICAL ACTIVITY OF
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MODELING THE BIOLOGICAL ACTIVITY OF
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LILIANA M. PĂCUREANU, ALINA BORA,
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A. R. ASHRAFI, P. NIKZAD, A. BEHMAR
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GHOLAM HOSSEIN FATH-TABAR, ALI REZA
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GHOLAM HOSSEIN FATH-TABAR, ALI REZA
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MODJTABA GHORBANI symmetric but it
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MODJTABA GHORBANI group can be comp
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MIRCEA V. DIUDEA, CSABA L. NAGY, PE
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STUDIA UBB. CHEMIA, LV, 4, 2010 WIE
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WIENER INDEX OF MICELLE-LIKE CHIRAL
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WIENER INDEX OF MICELLE-LIKE CHIRAL
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STUDIA UBB. CHEMIA, LV, 4, 2010 TUT
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TUTTE POLYNOMIAL OF AN INFINITE CLA
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ALI REZA ASHRAFI, HOSSEIN SHABANI,
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APPLICATION OF NUMERICAL METHODS IN
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APPLICATION OF NUMERICAL METHODS IN
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