Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.

Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.

Immunolocalization of water channel aquaporins in human knee articular cartilage with intact and early degenerative regions.

Aquaporins (AQPs), a family of water channel proteins expressed in various cells and tissues, serve as physiological pathways of water and small solute transport. Articular cartilage is avascular tissue with unique biomechanical structure, a major component of which is "water". Our objective is to investigate the immunolocalization and expression pattern changes of AQPs in articular cartilage with normal and early degenerative regions in the human knee joint, which is the joint most commonly involved in osteoarthritis (OA). Two isoforms (AQPs 1 and 3) of AQPs were examined by immunohistochemical analyses using isoform-specific antibodies with cartilage samples from OA patients undergoing total knee arthroplasty. AQP 1 and AQP 3 were expressed in human knee articular cartilage and were localized in chondrocytes, both in the intact and early degenerative cartilage regions. Compared to the intact cartilage, both AQP 1 and AQP 3 immunopositive cells were observed at the damaged surface area in the degenerative region. These findings suggest that these AQPs play roles in metabolic water regulation in articular cartilage of load bearing joints and that they are responsible for OA onset.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.

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 and localization of aquaporin 1b during oocyte development in the Japanese eel (Anguilla japonica).

To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica) novel-water selective aquaporin 1 (AQP1b) that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes.

[Expression of glutathione S-transferase-aquaporin 1 carboxyl terminal domain fusion protein in Escherchia coli].

We constructed a recombinant plasmid of water channel protein Aquaporin 1 (AQP1) carboxyl terminal domain (DNA sequence from 700bp-801bp) in pGEX-4T-1 vector and express the carboxyl terminal hydrophilic peptide AQP1 in E. coli. In this study, the DNA sequence of AQP1 hydrophilic peptide was amplified by PCR and was cloned into pGEX-4T-1 expression vector. After identified by restriction enzyme digestion and sequencing, the recombinant clone was transformed into the competent expression cells of E. coli BL21. The GST-AQP1 fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain a fusion protein with molecular weight of 30KD. So the fusion protein of AQP1 C-terminal hydrophilic peptide combined with GST was successfully expressed and purified. We set up important bases for the further research in AQP1 gene function.

Exceptional overproduction of a functional human membrane protein.

Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization.