Excess ligands were removed and buffer was exchanged into 0.25 M CH3COONH4 (pH 5.5) using a NAP-5 column (GE Healthcare, Glattbrugg, Switzerland).
Protein was concentrated, and buffer was exchanged into 10 mM Na2HPO4 and 500 mM NaCl.
Supernatants from baculovirus-infected High5 cells were concentrated on a stirred ultrafiltration cell (Amicon, Danvers, MA, USA) using YM-10 membranes (Millipore, Bedford, MA, USA), and buffer was exchanged into binding buffer (50 mM phosphate, 300 mM NaCl, and 10 mM imidazole, pH 7.4) on PD-10 desalting columns (GE Healthcare, Little Chalfont, UK).
The buffer of the purified protein fractions was exchanged into TEVp reaction buffer by using PD-10 desalting columns.
NsrR was exchanged into buffer C (50 mM Tris pH 7.0, 100 mM NaCl) to remove excess DTT.
The eluted protein was exchanged into buffer containing 50 mM Na Phosphate [pH 7.0], 250 mM NaCl, 1 mM EDTA, and 1 mM β-mercaptoethanol using an EconoPac PD10 desalting column (BioRad) and applied to a Superose 6 gel filtration column (GE Healthcare Life Sciences) equilibrated in the same buffer.
Protein was exchanged into buffer using three cycles of concentration-dilution with an Amicon Ultra-15 YM30 centrifugal filter.
The final protein was exchanged into buffer A (50 mM HEPES, pH 7.5, 0.3 M KCl, 1 mM DTT, 20% glycerol) using a Sephadex G-25 column, snap-frozen in small aliquots, and stored in liquid N2 until further use.
Prior to use Crm1 was exchanged into buffer G [50 mM HEPES-KOH, pH 7.5 @ 20°C, 50 mM KCl, 2 mM Mg(CH3CO2 2, 2 mM 2-mercaptoethanol, 2% (vol/vol) glycerol] using a 7 kD MWCO, Zeba Spin column (Thermo Scientific), and the concentration was determined using Bradford assays with a bovine serum albumin (BSA) standard (Thermo Scientific).
For in vitro biotinylation, N-CLCA1 containing the specific biotinylation tag at the C-terminus was exchanged into buffer B (100 mM Tris pH 7.5, 200 mM NaCl, and 5 mM MgCl2) and specifically biotinylated by addition of biotin and Escherichia coli BirA ligase (produced and purified in-house) at 4°C overnight.
Prior to conjugation the polymerase was exchanged into storage buffer without DTT either by dialysis or by using microSpin TM G-25 columns (Amersham Pharmacia Biotech).
