In the current study, X. laevis oocytes were used to express recombinant mammalian transport proteins for their subsequent purification and structural characterization. Channels and SLCs were taken as model proteins because they represent the majority of transport proteins, are linked to numerous inherited and acquired human diseases and correspond to key therapeutic targets. Purification was achieved by expressing recombinant proteins tagged with multiple epitopes and by using a novel procedure for the preparation of egg yolk-depleted total membranes. These two features were crucial for the successful purification of transport proteins. Five transport systems were purified in microgram amounts using the novel method: aquaporin-1, glutamate transporter 1, peptide transporter 1 and sodium-glucose-cotransporter 1 from human, and potassium-chloride cotransporter 4 from mouse. To validate our approach, we first tested the expression, localization and function of recombinant AQP1 and KCC4 in oocytes. Negative stain TEM and SPA of purified AQP1 and KCC4 indicated homogenous particle distributions and the expected oligomeric states. From the purification procedure described here, lastly, it was possible to grow 2D crystals of human AQP1 expressed in Xenopus laevis oocytes, paving the way for future structural analyses of mammalian membrane proteins by crystallography techniques. The structure determination of membrane proteins is lagging behind that of water-soluble proteins mainly due to the difficulty in heterologously expressing and isolating the required amounts for structural analyses. Although advances have been achieved over the past years, the number of structures of eukaryotic and in particular mammalian polytopic membrane proteins is still negligible. In the various cell types and systems that are currently used to express mammalian membrane proteins, i.e., bacteria, yeast, insect cells, mammalian cell lines and cell-free systems, proteins are often non-functional, mistargeted, misfolded, aggregated or degraded at abnormal rates. Yet, eukaryotic cell expression systems have been shown to be more appropriate than prokaryotic and cell-free systems to generate functional animal proteins because of their specific lipid environment and more elaborated translational and post-translational machineries. of an active transport system. Negative stain TEM and SPA showed highly homogeneous preparations of purified HA-AQP1 in the expected tetrameric form, implying correct protein folding and supramolecular assembly. On the other hand, KCC4 preparations were almost homogeneous, including a major population of larger particles and a minor one of smaller particles. The larger particles were at a size that is Vismodegib consistent with those of homodimers, which are one of the assembly forms taken by all KCC family members as suggested recently by biochemical experiments.