To purify the recombinant protein, the collected cell culture supernatant was centrifuged at 20,000g for 30 minutes at 4°C and prefiltered though a 0.2-μm polyvinylidene difluoride (PVDF) filter (Millipore, Bedford, MA). The resultant filtrate was diluted 1:1 (vol/vol) with binding buffer (20 mM Tris/HCl, 500 mM NaCl, 20 mM imidazole, 5 mM CaCl2 [pH 7.4]) and passed through a 5-mL Ni-chelate column (Amersham Pharmacia Biotech, Freiburg, Germany) using a commercial low-pressure liquid chromatography (LPLC) system (Äktaprime; Amersham Pharmacia Biotech). The bound proteins were eluted with 10 column volumes of a 0% to 50% linear gradient of elution buffer (20 mM Tris/HCl, 500 mM NaCl, 500 mM imidazole, 10 mM CaCl2 [pH 7.4]) in binding buffer. Collected fractions were tested by Western blot analysis, with antibodies against HisTag or myocilin (described later). Putative myocilin-containing fractions were pooled and dialyzed for at least 10 hours against 20 mM Tris/HCl, 100 mM NaCl, and 5 mM CaCl2 (pH 7.4), using a dialysis membrane with a 12- to 14-kDa cutoff (Spectra/Por; Spectrum Laboratories, Rancho Dominguez, CA).
For organ culture experiments, retentate from the above dialysis was further prepared by dialyzing it for at least 10 hours against the media used for perfusions (see later description) without added 1% FCS. The retentate was then ultracentrifuged (10,000g for 20 minutes at 4°C), and the protein content was measured (Bradford assay; Bio-Rad Laboratories, Munich, Germany). The sample was diluted with the dialysate (i.e., the solution outside the dialysis membrane) to give a protein concentration of 14 μg/mL. For experiments involving porcine eyes, 1% FCS was added to this solution, and for experiments with human eyes, 350 μg bovine serum albumin (BSA) was added. The control solution for organ culture experiments was the dialysate (with 1% FCS added for porcine perfusion or 350 mg/mL BSA added for human perfusion).