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Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells


X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI), and was recently used in our 2.1-Å X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of 0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8–10 weeks.

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Figure 1: Expression testing of full-length human Rh membrane proteins in transiently transfected HEK293S GnTI cells.
Figure 2
Figure 3: Dilution of transfected HEK293S GnTI cells before drug selection.
Figure 4: Expression levels from stably transfected and clonal HEK293S GnTI cell lines as determined by western blotting.
Figure 5: Medium- and large-scale HEK293S GnTI cell cultures.


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This work was supported by National Institutes of Health/National Institute of General Medical Sciences grants P50 GM73210, U54 GM094625 and R37 GM24485.

Author information




S.C., F.G. and R.M.S. designed the experiments. S.C. and F.G. performed the experiments. S.C., J.E.P., F.G. and R.M.S. analyzed the data. V.S. and R.M.S. supervised personnel. S.C., J.E.P. and R.M.S. wrote the paper.

Corresponding author

Correspondence to Robert M Stroud.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Full Sequence of pACMV-tetO. (DOC 27 kb)

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Chaudhary, S., Pak, J., Gruswitz, F. et al. Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat Protoc 7, 453–466 (2012).

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