RESUMEN
Aquaporin (AQP) is a water channel protein from the family of transmembrane proteins which facilitates the movement of water across the cell membrane. It is ubiquitous in nature, however the understanding of the water transport mechanism, especially for AQPs in microbes adapted to low temperatures, remains limited. AQP also has been recognized for its ability to be used for water filtration, but knowledge of the biochemical features necessary for its potential applications in industrial processes has been lacking. Therefore, this research was conducted to express, extract, solubilize, purify, and study the functional adaptations of the aquaporin Z family from Pseudomonas sp. AMS3 via molecular approaches. In this study, AqpZ1 AMS3 was successfully subcloned and expressed in E. coli BL21 (DE3) as a recombinant protein. The AqpZ1 AMS3 gene was expressed under optimized conditions and the best optimized condition for the AQP was in 0.5 mM IPTG incubated at 25°C for 20 h induction time. A zwitterionic mild detergent [(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate was the suitable surfactant for the protein solubilization. The protein was then purified via affinity chromatography. Liposome and proteoliposome was reconstituted to determine the particle size using dynamic light scattering. This information obtained from this psychrophilic AQP identified provides new insights into the structural adaptation of this protein at low temperatures and could be useful for low temperature application and molecular engineering purposes in the future.
Asunto(s)
Acuaporinas , Proteínas Bacterianas , Clonación Molecular , Escherichia coli , Pseudomonas , Proteínas Recombinantes , Pseudomonas/metabolismo , Pseudomonas/genética , Pseudomonas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Acuaporinas/química , Acuaporinas/genética , Acuaporinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Expresión Génica , Proteolípidos/metabolismo , Proteolípidos/química , Regiones Antárticas , Liposomas/metabolismo , Liposomas/química , Agua/química , Agua/metabolismo , Solubilidad , Secuencia de AminoácidosRESUMEN
The myelin and lymphocyte protein (MAL) family is a novel gene family first identified and characterized in 2002. This family is comprised of seven members, including MAL, MAL2, plasmolipin, MALL, myeloid differentiationassociated marker (MYADM), MYADML2 and CMTM8, which are located on different chromosomes. In addition to exhibiting extensive activity during transcytosis, the MAL family plays a vital role in the neurological, digestive, respiratory, genitourinary and other physiological systems. Furthermore, the intimate association between MAL and the pathogenesis, progression and metastasis of malignancies, attributable to several mechanisms such as DNA methylation has also been elucidated. In the present review, an overview of the structural and functional properties of the MAL family and the latest research findings regarding the relationship between several MAL members and various cancers is provided. Furthermore, the potential clinical and scientific significance of MAL is discussed and directions for future research are summarized.
Asunto(s)
Neoplasias , Proteolípidos , Humanos , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Proteolípidos/química , Proteolípidos/genética , Proteolípidos/metabolismo , Proteínas de la Mielina/genética , Proteínas , Neoplasias/genética , Transformación Celular Neoplásica , Carcinogénesis/genética , Linfocitos/metabolismo , Quimiocinas , Proteínas con Dominio MARVELRESUMEN
Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.
Asunto(s)
Membrana Dobles de Lípidos , Oxidación-Reducción , Proteolípidos , Proteolípidos/metabolismo , Proteolípidos/química , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/química , Cobre/metabolismo , Cobre/química , Hierro/metabolismo , Metales/metabolismo , Metales/química , Transporte Biológico , Liposomas Unilamelares/metabolismo , Liposomas Unilamelares/químicaRESUMEN
Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.