| 内容提要: |
Previous studies have shown that BsRS can be dissociated from the natural BsLS+BsRS encapsulation complex by changing the buffer conditions. To see if a similar buffer exchange would cause release of GFP11 from BsLS, I dialyzed one sample of purified BsLS+GFP11 complex (1 mL of a 1 mg/mL solution) in size-exclusion buffer(100 mM sodium phosphate, 1mM EDTA, pH 7.0) into 2 L 0.1 M sodium phosphate and 1 mM EDTA at pH 8.0 for 2 days, the other sample was dialyzed into 2L of pH 8.0 buffer containing 0.1 M Tris-HCl and 1 mM EDTA. Changing the dialysis buffer once after the first day. The sample was then subjected to analytical size-exclusion chromatography(HiPrep 16/60 Sephacryl S-400HR column) after passed through 0.22 µm syringe filter, as described above, using a running buffer containing 0.1 M sodium phosphate and 1 mM EDTA at pH 8.0 or 0.1 M Tris-HCl and 1 mM EDTA at pH 8.0. Fluorescence intensity at 508 nm and the absorbance at 280 nm of 200 µL sample were measured for each 4 mL fraction. To check for release of GFP11 with size-exclusion buffer (the standard working and storage buffer for the BsLS+GFP11 complex), 2 mL of freshly purified BsLS+GFP11 complex (3.5 mg/mL) was incubated at 4 °C in the dark for 28 days. An aliquot of this sample (0.57 mL) was mixed with size-exclusion buffer to a final volume of 2 mL, filtered, and then subjected to analytical size-exclusion chromatography (HiPrep 16/60 Sephacryl S-400HR column), as described above.Fluorescence intensity at 508 nm and the absorbance at 280 nm of 200 µL sample were measured for each 4 mL fraction. The fluorescence intensity was measured by Tecan Infinite M200 PRO plate reader, then transfer the sample from the 96-well plate to cuvette to measure the absorbance at 280 nm using an Eppendorf Biophotometer. |