Eluted proteins underwent buffer exchange and were concentrated using Centricon purifying units (Millipore) with 10-kDa (for TAXGST-CNB-1) or 50-kDa (for other proteins) cutoffs to 0.5 1.0 mg/ml protein in storage buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5% NP-40NP-40, 10cerol); the resulting solutions contained >1 mM glutathione or >5 mM imidazole.
The purified enzyme in TE buffer (pH 7.5) was concentrated by using 30-kDa cutoff Centrifugal filter device (Ultracel YM-30, Millipore, Billerica, MA, USA).
Following overnight incubation, the chitin/resin slurry was returned to the column and the liberated semisynthetic enzyme was eluted via gravity flow by further washing of the resin with chitin buffer B. The semisynthetic enzyme was concentrated and buffer exchanged with buffer C [10 mM Tris-EDTA, 10 mM NaCl, and 20 mM βME (pH 8.0)] via ultrafiltration (Amicon Ultracel 30 kDa cutoff filters).
The mitochondrial pellet was lysed in Laemmli buffer, and the cytosolic supernatant was concentrated with a Microcon device (size cut-off 10 kDa) (Millipore Corporation, Bedford, MA, USA).
Whole-cell lysates were collected with radioimmunoprecipitation assay buffer, and medium from cells was concentrated using a centrifugal filter unit (EMD Millipore, Billerica, MA, USA).
To concentrate and exchange the buffer, purified CAHL-C was concentrated up to 40 times with PBS by using Amicon Ultra 30 (Millipore, EMD, Germany).
The MBP tag was then utilized to purify the protein on an amylose column, and the protein was concentrated and exchanged into buffer C (25 mM HEPES-KOH pH 7.4, 100 mM NaCl, 10% glycerol (v/v)).
The 4 L of refold buffer containing the rCR2 was concentrated to ∼50 mL by ultrafiltration using a Vivaflow 200 5 kDa membrane cutoff tangential flow filtration system (Sartorius Stedim, Surrey, UK).
Fractions containing the recombinant protein (showing a band of the expected size on a SDS-PAGE gel) were pooled, dialyzed against buffer A. The protein was concentrated to 2 mL using a Amicon Ultra centrifugal filter device (Millipore, USA), applied to a Sephacryl S-200 gel filtration column (Pharmacia, USA) pre-equilibrated with buffer A and eluted with the same buffer.
