Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1)

Abstract

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.

Fig 1. PCFT expression in Sf9 cells.

A. Sf9 cells in 50 ml suspension culture at a density of 2 x 10⁶ cells/ml were infected at a MOI of 2. One-ml whole cell samples were collected at the indicated time points, electrophoresed on a 4–15% Mini Protean TGX Precast SDS-PAGE gel (BioRad), transferred to a PVDF membrane, and immunoblotted using an antibody against the His₆ tag of PCFT. PCFT bands were observed at ~39 and ~43 kDa. The highest PCFT expression was observed 48 h post infection. No PCFT expression was observed at the time of infection (0 h) or in uninfected cells after 48 h (uninf). B. Treatment of membrane vesicles with PNGase F under non-denaturing conditions shifted the PCFT band from ~43 kDa to ~39 kDa (each lane treated with PNGase corresponds to a different sample preparation).

Fig 2. Detergent screening for solubilization of PCFT.

Nine different detergents were used at the indicated concentrations to analyze solubilization of PCFT from membranes isolated from Sf9 cells. After a 2-h incubation, solubilized supernatants and pellets were analyzed using 4–15% MiniProtean TGX Precast SDS-PAGE gels, transferred to PVDF membranes and immunoblotted for detection with an antibody against the His₆ tag of PCFT. First lane (Total protein) is the total amount of PCFT in Sf9 crude membranes before solubilization, and the second lane is a sample without detergent. (A) Non-solubilized pellets of the initial screen, (B) solubilized supernatants of the initial screen, and (C) solubilized supernatants with increased OG and CHAPS concentrations. See text for detergent abbreviations.

Fig 3. Lipid reconstitution of PCFT.

PCFT was reconstituted in liposomes as described in Experimental Procedures. Purified protein and reconstituted PCFT were subjected to SDS-PAGE (4–15% MiniProtean TGX Precast gel). (A) Protein staining (Stain-free gel, BioRad) and (B) Western blot of the same gel analyzed using an antibody against the His₆ tag of PCFT. Lane 1: purified protein eluted from the Talon Co²⁺ resin; lane 2: PCFT reconstituted in proteoliposomes.

Fig 4. Size-exclusion chromatography analysis of PCFT.

(A) Elution profile of PCFT solubilized in DDM. Elution volumes of standard proteins are indicated as follows: thyroglobulin (T, 669 kDa), ferritin (F, 440 kDa), aldolase (A, 158 kDa), conalbumin (C, 75 kDa), ovalbumin (O, 44 kDa). Blue dextran (BD, 2 MDa) was used for void volume determination. (B) PCFT fraction corresponding to the peak PCFT elution fraction was analyzed by protein staining (BioRad stain-free imaging). (C) The partition coefficients (K_av) of the standard proteins are plotted against the log of their molecular weights to calculate the size of the PCFT-DDM complex, yielding an apparent size of 280 kDa.

Fig 5. PCFT-dependent ³H-folic acid uptake in Sf9 cells.

(A) 10-min uptake of 500 nM ³H-folic acid (FA) by PCFT-expressing Sf9 cells determined at pH 5.5. Data are means ± SD. The value in Sf9 cells expressing PCFT (PCFT) was significantly different from that of uninfected cells (Control) (1-way ANOVA with Tukey’s multiple comparison test, P ≤ 0.0001, ****). Uptake was reduced significantly in the presence of a 200-fold excess of unlabeled folic acid (PCFT + Ex FA) (1-way ANOVA with Tukey’s multiple comparison test, P ≤ 0.0001, ****). The difference between the PCFT + Ex FA and Control was not significant (ns). (B) Concentration dependence of the ³H-folic acid uptake in PCFT-expressing Sf9 cells (PCFT) and uninfected cells (Control). Data was fit using the Michaelis Menten equation (Graphpad Prism, San Diego, CA).

Fig 6. ³H-folic acid uptake by purified PCFT reconstituted in liposomes.

The 30-s uptake of 300 nM ³H-folic acid (FA) into unilamellar PCFT-proteliposomes (PCFT-PL) was measured at pH 5.5. Unilamellar empty liposomes (L) served as control. The uptake in PCFT-PL was significantly higher than that in liposomes (t-test: L–PCFT-PL P = 0.008, r² = 0.86; one preparation, n = 3).