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. 2011 Dec;55(4):273-80.
doi: 10.1016/j.ymeth.2011.08.018. Epub 2011 Sep 8.

Efficient Expression Screening of Human Membrane Proteins in Transiently Transfected Human Embryonic Kidney 293S Cells

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Efficient Expression Screening of Human Membrane Proteins in Transiently Transfected Human Embryonic Kidney 293S Cells

Sarika Chaudhary et al. Methods. .
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It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.


Figure 1
Figure 1. Two-step PCR schematic
Individual PCR steps are enclosed in blue boxes and labelled as PCR I and PCR II. The primers are labelled as 3C, Thrombin, FLAG, and 10x HIS for simplicity. The image is not drawn to scale. See the text and Table 1 for details.
Figure 2
Figure 2. Comparison of membrane protein expression levels in the presence and absence of spacer sequences
A) Five stably transfected HEK 293S clonal cell lines (labelled as 1 to 5) expressing a full length human solute carrier and B) a stably transfected HEK 293S clonal cell line expressing a full length human receptor, resolved on reducing SDS-PAGE and analyzed via Western blotting. In both cases, (-) and (+) denote the absence and presence of a 5 amino acid spacer sequence at the C-terminus of the membrane protein, respectively (see text for details). BS and AS denote “before spin” and “after spin” samples (see section 2.3 for details). Membranes were probed using an anti-FLAG-HRP monoclonal antibody (1:1000 dilution).
Figure 3
Figure 3. pACMV-tetO-EGFP expression vector
An arbitrary transgene (yellow arrow) has been inserted into the vector at the appropriate location, N-terminal to the EGFP molecule (green arrow) using the KpnI and NotI restriction sites, for illustrative purposes.
Figure 4
Figure 4. Western blotting analysis of small-scale, whole cell solubilized human membrane proteins
Three full-length human membrane proteins, subcloned into pACMV-tetO-EGFP, were solubilized from GnT I −/− HEK 293S cells (in this case stably transfected) and resolved on a reducing SDS-PAGE. Western blotting was performed using a 1:1000 dilution of anti-His-HRP and a film exposure time of 20 seconds. BS and AS denote the “before spin” and “after spin” samples (see text for details).
Figure 5
Figure 5. Example of using FSEC to assess expression levels quantitatively
A. Examples of FSEC traces from a mammalian transporter. The asterisk denotes the included peak used for assessment of expression levels. Absorbance at 280 nm (A280) for each construct is depicted below the FSEC traces. B. Relative expression levels normalized to the A280 trace for the three constructs. For each construct, five independent transfections where performed and the normalized expression level was calculated. The asterisk denotes the transfection experiment that gave the chromatograms shown in panel A. For this mammalian transporter, the full length construct did not have an included peak and was discarded. All three truncated constructs produced improved, included FSEC peaks of comparable quality. Based on the FSEC profiles shown in panel A, construct 2 might be expected to have the highest expression. Nevertheless, analysis of the normalized expression levels (panel B) reveals that construct 1 in fact possess the highest expression.
Figure 6
Figure 6. Fluorescence-detection size-exclusion screening of human membrane proteins solubilized from HEK 293S cells
100 μL of solubilized full-length human membrane proteins (see Figure 4) were injected onto a TSK-G3000SW column equilibrated in 50 mM Hepes pH 7, 200 mM NaCl, and detergent (0.5 mM β-DDM, 0.5 mM FC 14, 40 mM β-OG) at a flow rate of 0.5 ml/min. Fluorescence detection was monitored as described in section 2.4. The void volume, included volume and free EGFP molecule are labelled for the center chromatogram.
Figure 7
Figure 7. Comparison of human membrane proteins expressed in HEK 293S and SF9 cells
A human solute carrier family member of approximately 60 kDa was expressed in stably transfected GnT I −/− HEK 293S and Sf9 cells. In both cases, whole cell membrane solubilized material (using 1% β-DDM) was resolved on a reducing SDS-PAGE, and immunoblotted using an anti-FLAG (HRP) monoclonal antibody. For the HEK 293S expression trials, each lane corresponds to a clonal cell line stably transformed with transgene subcloned into pACMV-tetO. For Sf9 expression, the transgene was expressed using a pFASTBac expression vector (Invitrogen, 10712024), followed by a P2 viral dilution of either 1:200, or 1:500 for infection. In the case of the Sf9 produced material, both higher order aggregates (>250 kDa) and degredation products (20 kDa) were observed, in addition to the expected 60 kDa band, suggesting that Sf9 may not be suitable for the expression of this human solute carrier family member. While higher order aggregates (~150 kDa) were also observed in HEK-293S, their magnitude relative to the 60 kDa band appeared to be less for some clonal cell lines (see clones 4,6 and 9) relative to that observed for Sf9 (see 1:200 diultion).

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