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

Abstract

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.

Figure 1

Expression testing of full-length human Rh membrane proteins in transiently transfected HEK293S GnTI- cells. 10 μg of RhAG, RhBG and RhCG, subcloned into pACMV-tetO, were separately transfected into HEK293S GnTI- cells grown in 10 cm² tissue culture plates. Cells were induced with doxycycline (24 hours post transfection), harvested (24 hours post induction), and analyzed via anti-His Western blotting, as described in the text. Based on these results, RhAG and RhCG were chosen as candidates for stable cell line generation.

Figure 2

Workflow for the overexpression of mammalian membrane proteins in stably transfected and clonal HEK293S GnTI- cells.

Figure 3

Dilution of tranfected HEK293S GnTI- cells prior to drug selection. Cells from one well of a 6 well tissue culture plate (2 mL total) are diluted into five 10 cm² tissue culture plates, by plating the appropriate amount of cells (indicated in the boxes). Each 10 cm² plate possess 10 mL of DMEM. The final dilution factor is indicated for each plate.

Figure 4

Expression levels from stably transfected and clonal HEK293S GnTI- cell lines as determined by western blotting. Each lane corresponds to the “after spin” sample from a separate clonal cell line (clones 1 to 9), visualized via anti-FLAG western blotting. All clones were solubilized using DDM. See text for experimental details.

Figure 5

Medium and large scale HEK293S GnTI- cell cultures. As shown in panel A, up to 7 × 1 L spinner flasks can be maintained in a single CO₂ incubator. Alternatively, up to 3 × 3 L spinner flasks can maintained (not shown). In panel B, 2 × 10 L WAVE bioreactors, using cellbags manufactured by GE Healthcare (left) and Flex Concepts Inc. (right), are shown. Relevant features of the cellbag and WAVE bioreactor, as discussed in the text, are highlighted.