Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

Abstract

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Expression, Purification and Low-Resolution Structure of Human Vitamin C Transporter SVCT1 (SLC23A1)

“…cDNA from hSVCT1 (UniProtKB entry: Q9UHI7) was cloned by PCR from a carrier construct of our laboratory into the pMJB08 expression vector [19]. The forward primer 5′ GGG GAA TTC ATG AGG GCC CAG GAG GAC CTC 3′ and the reverse primer 5′ GGG AAG CTT TCA GAC CTT GGT GCA CAC AGA TGC 3′ were used.…”

“…The forward primer 5′ GGG GAA TTC ATG AGG GCC CAG GAG GAC CTC 3′ and the reverse primer 5′ GGG AAG CTT TCA GAC CTT GGT GCA CAC AGA TGC 3′ were used. PCR products were digested with the restriction enzymes EcoRI and HindIII, and ligated into the pMJB08 vector [19]. The DNA construct was verified by sequencing.…”

“…a hemagglutinin (HA) epitope. For more details on pMJB08 see Bergeron et al [19]. The calculated molecular masses of recombinant hSVCT1 based on the amino acid sequence are ∼70.8 kDa (full-length) and ∼67.2 kDa after Prescission protease cleavage.…”

“…Expression of hSVCT1 was performed as previously described for other mammalian and human transporters by Bergeron et al (2011) [19]. This methodological report from our laboratory also describes in great detail the used procedure for the isolation of oocyte membranes containing expressed hSVCT1 and the subsequent affinity purification.…”

“…For crosslinking, membranes from 1,000 oocytes were isolated as previously described [19], but using 20 mM HEPES-NaOH (pH 7.5) instead of 20 mM Tris-HCl (pH 8) in all buffers. This is necessary because Tris contains a primary amine that competes with amine-reactive crosslinkers such as DST.…”