 | Acta Crystallographica Section D Acta Crystallographica Section D BIOLOGICAL CRYSTALLOGRAPHY |
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![[Figure 4]](gx5088fig4mag.jpg)
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Figure 4
Calibration tests for protein expression in eukaryotic cells. All panels are Western blots probed with an anti-His-tag monoclonal antibody (see § ![[link]](../../../../../../logos/arrows/d_arr.gif) 2.6). (a) Small versus large-scale expression: three constructs were transfected either in six-well dishes (lanes 1, 3 and 5) or in roller bottles (lanes 2, 4 and 6) and samples of conditioned media collected 3 d later. A higher concentration of protein in the conditioned media was obtained in the small-scale experiments, but the ratios between small-scale and large-scale yields appear constant, thus allowing a good estimate of the scale-up required. (b) Expression of a large (170 kDa) membrane protein in HEK293T (lanes 2–4, transient transfection) and Sf9 (lanes 5–7, baculovirus infection) cells. Lane 1 is negative control (non-transfected HEK293T cells). Samples containing similar amounts of cells were collected 24 h (lanes 2 and 5), 48 h (lanes 3 and 6) and 72 h (lanes 4 and 7) post-transfection/infection. The 200 kDa band observed in the HEK cells samples arises from more complex glycosylation. A breakdown product of ∼120 kDa becomes apparent as cultures grow older. (c) Parallel expression test of two intracellular proteins in HEK293T (lanes 2 and 3) and Sf9 (lanes 4 and 5) cells. Lanes 1 and 6 are negative controls (non-transfected/non-infected HEK293T/Sf9 cells, respectively). One construct (lanes 2 and 4) is poorly expressed in both systems, while the second construct (lanes 3 and 5) appears to be better expressed in insect cells. (d) SeMet addition to the tissue-culture media, post-transfection, causes a reduction in protein yield. Lane 1, complete medium; lane 2, Met-free medium supplemented with 30 mg l−1 L-methionine; lanes 3, 4 and 5, Met-free medium supplemented with 30 mg l−1 SeMet (three different batches). |
![[Figure 4]](gx5088fig4.jpg)
| BIOLOGICAL CRYSTALLOGRAPHY |
ISSN: 1399-0047
