Preparative scale production of functional mouse aquaporin 4 using different cell-free expression modes

Abstract

The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated P(f) value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.

Figure 1. Resolubilization screening of P-CF produced mAQP4 M23.

The pellet from the P-CF reaction mix was resuspended with either 1% (w/v) Fos-12, 2% (w/v) DHPC, 2% (w/v) Fos-16, 2% (w/v) LMPG, or 1% (w/v) LPPG. Sample volumes of 4 µl were analyzed by 16% SDS-PAGE. The solubilization efficiency was determined by densitometry after immunoblotting using anti-His antibodies. Control is P-CF expressed mAQP4 M23. A: immunoblotting using anti-His antibodies. S, supernatant; P, pellet. B: The solubilization efficiency determined by densitometry of the immunoblotting.

Figure 2. Detergent screening of mAQP4 M23 expressed in the D-CF mode.

A: RM samples of 2 µl were analyzed by 16% SDS-PAGE and immunoblotted using anti-His antibodies. B: Solubility of D-CF expressed mAQP4 M23 in presence of 0.2% Brij-35, 0.4% Digitonin, 0.1% Triton X-100, and 0.05% Tyloxapol. Control is P-CF expressed mAQP4 M23. S, supernatant; P, pellet.

Figure 3. Purification of D-CF produced mAQP4 M23 in 0.2% Brij-35 by Co²⁺-NTA chromatography.

Samples were separated by 12% (lanes 1–2) or 16% (lanes 3–7) SDS-PAGE and analysed by Coomassie staining. Lanes 1 and 3, protein marker; Lane 2, precipitate after P-CF expression; Lane 4, flow through; Lane 5, washing fraction; Lane 6, elution fraction; Lane 7, immunoblot of lane 6 using anti-His antibodies. Samples of 2 µl were applied to each lane.

Figure 4. Water transport activity of P-CF and D-CF mode produced mAQP4 M23.

Precipitates of P-CF produced mAQP4 M23 were resolubilized in the indicated detergents. The solubilized proteins were purified by Co²⁺-NTA chromatography, the initial detergent exchanged to 0.05% DDM and the samples were reconstituted into E. coli polar lipids. Water transport activity was determined by stopped-flow light scattering measurements of mAQP4 M23 proteoliposomes at 10°C. A 200 mM osmotic gradient was established by rapidly mixing vesicles suspended in reconstitution buffer with an equal volume of reconstitution buffer +400 mM sucrose. Data represent the average of three independent measurements. Fitted curves of mAQP4 M23 proteoliposome light scattering are shown. Solid line, mAQP4 M23 proteoliposomes; Dashed line, E. coli polar lipid empty liposomes.

Figure 5. Specific inhibition of mAQP4 M23 water transport.

Proteoliposomes containing D-CF produced mAQP4 M23 were treated with 300 µM HgCl₂ for 5 min. at 23°C. For the recovery of the function of mAQP4 M23 function, 2 mM β-mercaptoethanol (β-ME) was added and incubated 10 min. at 23°C after incubation with HgCl₂. Empty liposomes with and without treatment by HgCl2 were used as control and showed identical curves (dashed line).

Figure 6. Flow-chart of mAQP4 M23 production by CF expression.

The complete process from expression to functional analysis is finished within 2 days.