Sepharose

Example 9

NEBT7EL-pA06238 was grown on LB with 50 μg/ml kanamycin. A 600 ml culture of TB_kan50 was inoculated with NEBT7EL-pA06238 and incubated overnight at 37° C. at 200 rpm. The next morning, a 10 L fermentor was prepared with 9.5 L of TB and then inoculated with 500 ml of the overnight culture. The culture was grown at 37° C. The pH was maintained at 6.2 with NaOH and the dO₂ was maintained ≥20%. After 2 hours of growth, the temperature was dropped to 25° C. The culture was grown for an additional 1 hour with the OD₆₀₀ around 7. IPTG was added to a final concentration of 1 mM and CoCl₂ was added to 25 μM. Additional CoCl₂ was added 1 and 2 hours after induction to bring the final concentration to 300 μM. The cells were grown for 20 hours at which point the fermentor was chilled to 10° C. and the cells were harvested by centrifugation. The cell pellet was stored at −80° C. until use.

The cell pellet from the fermentation was lysed by stirring in buffer with lysozyme and DNAse. Cell debris was removed by centrifugation and the supernatant was filtered through a 0.45 micron filter. Filtered supernatant was incubated with Ni-NTA agarose resin and then enzyme was eluted with imidazole. Purified FC4E pA06238 was immobilized onto 5.25 grams of ECR8204F resin using the standard published protocol from Purolite.

The immobilized enzyme was loaded into a 11×300 mm glass fixed bed reactor and run for approximately 200 h at constant temperature (60° C.) with a constant feed composition of 30 wt % fructose+70 wt % aqueous buffer solution (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂). Feed rate was held constant at 140 uL/min throughout the run. The fixed bed reaction reached a maximal conversion of approximately 30% tagatose and had a half-life of −50 hours (FIG. 15).

Example 2

About 5 μM fluorescein (F1300, Invitrogen, Carlsbad, CA) solution in ethanol was prepared. For imaging, the solution was transferred into a sealed 10 mm glass bottom dish (P35G-1.5-10-c, MatTek Corporation, Ashland, MA, USA) and mounted in an inverted confocal microscope. Imaging was performed on a Zeiss LSM780 inverted confocal microscope with QUASAR detector (Carl Zeiss, Jena, Germany). A typical dataset consists of 32 images, each of dimensions 512×512 pixels, corresponding to different wavelengths from about 410.5 nm to about 694.9 nm with about 8.9 nm bandwidth. The measurement is repeated 10 times using C-Apochromat 40×/1.20 W Korr Zeiss objective at any given imaging parameter. Fluorescein was imaged with about 488 nm laser at different acquisition parameters (Table 1).

For in vivo imaging 5-6 zebrafish embryos at appropriate stage were placed into about 1% agarose (Catalog No. 16500-100, Invitrogen™) moulds created in an imaging dish with #1.5 coverglass bottom, (Catalog No. D5040P, WillCo Wells) using a custom designed negative plastic mould [29]. Embryos were immobilized by adding about 2 ml of about 1% UltraPure™ Low Melting Point Agarose (Catalog No. 16520-050, Invitrogen™) solution prepared in about 30% Danieau (about 17.4 mM NaCl, about 210 μM KCl, about 120 μM MgSO₄.7H₂O, about 180 μM Ca(NO₃)₂, about 1.5 mM HEPES buffer in water, pH about 7.6) with about 0.003% PTU and about 0.01% tricaine. This solution was then added on top of the embryos already placed in the mold. Following solidification of agarose at room temperature (1-2 minutes), the imaging dish was filled with about 30% Danieau solution and about 0.01% Tricaine, at about 28.5° C. Subsequent imaging was performed on an inverted confocal microscope by positioning the petridish appropriately on the microscope stage. Samples were obtained by crossing Gt(desm-citrine)^ct122a/+ with Tg(kdrl:eGFP) fish for two color imaging. Samples with four fluorescent proteins result from same crossing followed by injection of about 100 pg per embryo of mRNA encoding H2B-cerulean and membrane-mCherry. Samples of Gt(desm-citrine)^ct122a/+;Tg(kdrl:eGFP) were imaged with about 488 nm laser to excite both Citrine and eGFP and a narrow about 488 nm dichroic to separate excitation and fluorescence emission. Samples of Gt(desm-citrine)^ct122a/+;Tg(kdrl:eGFP) with H2B-cerulean and membrane-mCherry labels were imaged with about 458 nm laser to excite Cerulean, eGFP and Citrine with a narrow about 488 nm dichroic, following an about 561 nm laser to excite mCherry with an about 458-561 nm dichroic.

For in vivo time-lapse imaging 5-6 zebrafish at appropriate stage were immobilized in an imaging dish with #1.5 coverglass bottom using about 0.5% Low Melting Point Agarose agarose (same as above) to allow for development and with about 0.003% PTU and about 0.01% tricaine. Subsequent imaging was performed on the same confocal-two photon inverted microscope at about 28.5° C. A solution of Egg Water was added every hour to the imaging dish to ensure proper hydration of the sample. Samples with five fluorescent proteins were obtained by crossing Tg(kdrl: eGFP) with Tg(ubiq:membrane-Cerulean-2a-H2B-tdTomato) zebrafish followed by injection of about 120 pg and about 30 pg per embryo of mRNA encoding Rab9-YFP and Rab11-mCherry, respectively. Volumetric data was acquired using about 950 nm to excite Cerulean, eGFP, YFP and (weakly) tdTomato with a 760+ bandpass filter, following an about 561 nm laser to excite mCherry and tdTomato with an about 458-561 nm dichroic.

Table 3 provides the detailed description of the imaging parameters used for all images presented in this work.

Example 3

Cultivations were harvested on day 14 by centrifugation at 8000 g for 40 min. Filtered supernatants were purified for further characterization of IL-2 mutein Ala-M1. The supernatant was concentrated and loaded onto a Superdex 75 prep grade column (GE Healthcare Life Sciences, now Cytiva) equilibrated in 25 mM Tris, 200 mM NaCl pH 8.0. Fractions were analyzed by SDS-PAGE and fractions containing target protein were pooled. Further purification was done by cation exchange chromatography on an SP Sepharose HP column (GE Healthcare Life Sciences, now Cytiva) equilibrated in 25 mM Na-acetate pH 5.5. Before loading, the protein pool was pH adjusted to 5.5 and diluted with water. Elution was done by linear salt gradient elution. Fractions were analyzed by SDS-PAGE and those containing target protein were pooled to give purified IL-2 mutein Ala-M1 1 (SEQ ID NO:14 with an additional cysteine or glutathione connected to the thiol group of the cysteine at position 38 via a disulfide bridge).