pVT-mdr3.5

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	Transformation of P. pastoris strain KM71H and expression analysis were as previously described [31], [35]. Selected strains were grown in a BioFlow IV fermentor and the proteins purified as previously described [13] with the following modifications: 10 mM DTT was included during cell breakage in a glass bead beater to fully reduce the proteins, and all buffers for membrane preparation and chromatography were supplemented with 1 mM β-mercaptoethanol and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) to keep proteins reduced.

Protocol tips

Transformation of P. pastoris strain KM71H and expression analysis were as previously described [31], [35]. Selected strains were grown in a BioFlow IV fermentor and the proteins purified as previously described [13] with the following modifications: 10 mM DTT was included during cell breakage in a glass bead beater to fully reduce the proteins, and all buffers for membrane preparation and chromatography were supplemented with 1 mM β-mercaptoethanol and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) to keep proteins reduced.

Publication protocol

Transformation of P. pastoris strain KM71H and expression analysis were as previously described [31], [35]. Selected strains were grown in a BioFlow IV fermentor and the proteins purified as previously described [13] with the following modifications: 10 mM DTT was included during cell breakage in a glass bead beater to fully reduce the proteins, and all buffers for membrane preparation and chromatography were supplemented with 1 mM β-mercaptoethanol and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) to keep proteins reduced. Proteins were concentrated to approximately 1 mg/ml using YM-100 Ultrafilters (Millipore). The concentrated protein was aliquoted and stored at −80°C. For gel filtration chromatography, protein was concentrated to 4 mg/ml and 0.5 ml chromatographed on Superose 6B (10×300 mm, GE Healthcare) in 20 mM Hepes-NaOH pH 7.4, 10% glycerol, 50 mM NaCl, 1 mM DTT and 0.2% n-Dodecyl-β-D-maltopyranoside (DDM) using an Äkta Purifier chromatography system (GE Healthcare). Pgp concentrations were routinely determined by UV spectroscopy at 280 nm using a calculated extinction coefficient of 1.28 per mg/ml. Serial dilutions of WT- and Opti-Pgp preparations were further assayed side-by-side with the colorimetric BCA protein assay (Pierce) using BSA with appropriate buffer controls as a standard; the two assays gave essentially the same results. Finally, increasing concentrations of different protein preparations were resolved side-by-side on Coomassie-stained SDS-gels, individual lanes were scanned and the amount of protein in the Pgp and other protein bands quantitated using ImageJ (http://rsbweb.nih.gov/ij/). The latter method permits visual inspection as well as quantitative validation of samples and allows for direct comparison of the Pgp content of the samples.

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