 | Acta Crystallographica Section D Acta Crystallographica Section D BIOLOGICAL CRYSTALLOGRAPHY |
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![[Figure 1]](gx5098fig1mag.jpg)
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Figure 1
Representative experiments of total expression (a) and soluble expression after IMAC purification (b) from Utrecht using RosettaII (DE3), as determined by SDS–PAGE. (c) Soluble expression from Oxford after IMAC in Rosetta (DE3)-pLysS as determined by SDS–PAGE. The first and last lanes are molecular-weight markers. (d) Insoluble (pellet) and soluble expression from Stockholm after IMAC in the indicated bacterial strains BL21 (DE3) or RosettaII (DE3). Protein was detected using the dot-blot procedure described by Knaust & Nordlund (2001  ). The scale on the right is displayed as an indicator for `very good', `good', `weak', `poor' and `non' expressed protein. (e) Soluble expression as determined by Marseille using a dot-blot procedure (Vincentelli et al., 2005), where expression was performed in parallel (1152 conditions) using three different media (light grey, 2YT; dark grey, SB; black, TB), three different culture temperatures (290, 298 and 310 K) and four different bacterial strains [B = BL21 (DE3)-pLysS, R = Rosetta (DE3)-pLysS, O = Origami (DE3)-pLysS, C = C41 (DE3)-pLysSRARE]. 12 out of the 36 possible conditions were used in an incomplete factorial approach as schematically indicated in the shaded table [e.g. the first spot of every expression test refers to an experiment performed at 310 K in BL21 (DE3)-pLysS using 2YT]. The results for standard amounts spotted for referencing are shown alongside (left column from top to bottom, 2000, 1500, 1000, 900, 800, 700, 600 and 500 ng per dot; right column from top to bottom, 400, 300, 200, 100, 50, 25, 12.5 and 0 ng per dot). The quantification is performed automatically with the microplate reader implemented on the Tecan robot (photon, calibration curve). Molecular-weight markers in kDa are indicated. |
![[Figure 1]](gx5098fig1.jpg)
| BIOLOGICAL CRYSTALLOGRAPHY |
ISSN: 1399-0047
