Table 1.
Alphabetical list of the CWGTs that were screened.
Fig 1.
Flow diagram of the high-throughput expression screening pipeline.
Table 2.
Hits from the test library screening.
Fig 2.
The effect of salts, pH and additives in the lysis buffer on the solubility of RGP1.
Western blots against a cloning scar shows the varying amount of RGP1 in the soluble lysis fraction after lysing in different buffers. The arrow indicates the band corresponding to full-length RGP1 (42 kDa). Buffers 13 and 19–21, containing 1-2M salt, as well as the low-pH (4.3–5.5) buffers 11,12,15, 22 and 30 decrease the amount of soluble RGP1, while lower salt concentrations as well as several additives increase the amount of soluble RGP1 relative to the standard lysis buffer (std: 50 mM HEPES pH 8.0, 250 mM NaCl, 5 mM MgCl2). MSG = monosodium glutamate, Dex = dextran. *Condition 24: 100 mM triethanolamine, 80 mM sodium glutamate, 0.02% n-octyl-β-D-glucoside, 10% glycerol, 5mM MgSO4, pH 8.5. **Condition 29: 100 mM Tris, 100 mM KCl, 0.1% deoxycholate, 25% glycerol, pH 7.6. ***Condition 30: 100 mM potassium acetate, 50 mM NaCl, 0.05% dextran sulfate, 0.1% CHAPS, pH 5.5.
Table 3.
Hits from the non-Arabidopsis library screening.
Fig 3.
SDS-PAGE of selected samples from the non-Arabidopsis library.
Coomassie-stained gels showing nickel affinity eluates of samples selected from the automated capillary electrophoresis procedure as described. Each lane is marked with the organism and protein name, as well as with the well number for reference to S6 Data. Bands that were excised for mass spectrometry are marked with a green dot (confirmed by mass spectrometry) or a red cross (not confirmed). The lower right gel has samples expressed in the periplasm (pET22DEST vector, no excisions), while the other three gels have samples expressed in the cytoplasm (pET55DEST vector). Sample b8 (upper left gel) has no detectable background on SDS-PAGE, but produces a normal band pattern in the automated capillary electrophoresis, indicating that the absence of bands on SDS-PAGE is likely due to faulty well loading. Ca = Cicer arietinum, Cs = Cucumis sativa, Fv = Fragaria vesca, Gm = Glycine max, Si = Setaria italica, Sl = Solanum lycopersicum, Vv = Vitis vinifera, Zm = Zea mays.
Fig 4.
Arabidopsis thaliana RGP1 scale-up, purification and activity determinations.
(A) Coomassie stained SDS-PAGE of the final chromatographic step yielding almost pure RGP1 (42 kDa). (B) Phosphate-release assay showing autoglycosylating or hydrolytic activity of RGP1 on UDP-glucose. (C) UDP-arabinose mutase activity of RGP1. High-pressure liquid chromatograms of authentic UDP-arabinopyranose (UDP-Arap) and UDP-arabinofuranose (UDP-Araf) standards (grey) overlaid with the chromatogram of the reaction mixture of UDP-Araf with recombinant, purified RGP1 (black).