Recombinant production and characterization of aquaporin protein isolated from geobacillus thermoleovorans ARTR1 and virgibacillus sp. agtr strains

Abstract

Cells have a fluid mosaic model that provides the passage of organic and inorganic molecules, ions and water, which are survival for the cell, that form the structure of cell membranes and contain protein, carbohydrates and cholesterol. Proteins located in the membrane of the cell are called biological membrane proteins and they constitute approximately 25-30 % of all proteins. However, the partially hydrophobic surfaces of membrane proteins are still a largely unconquered area due to their lack of flexibility and stability. Biological membrane proteins have three subgroups depending on their location are analyzed in category. Integral membrane proteins, one of the biological membrane proteins which are permanently attached to the cell membrane. Detergents, non-polar solvents, or sometimes denaturing agents are used to separate this protein from the biological membrane. Integral membrane proteins are a permanent part of the membrane and can penetrate the membrane to form transmembrane proteins. Peripheral membrane proteins, another biological membrane protein, temporarily bind to the membrane or integral membrane proteins by hydrophobic, electrostatic, and other non-covalent interactions. These proteins can dissociate from the membrane after treatment with a solution containing a high pH or high salt concentration. Lipid-anchored proteins, on the other hand, are proteins on the surface of the cell membrane that covalently bind to lipids embedded in the cell membrane. Today, the structural and functional studies of the above-mentioned biological membrane proteins have been largely hampered by the difficulties in their production and overexpression, which are necessary for the structural studies of membrane proteins. Compared to other protein classes, the determination of the structure, expression, and purification of membrane proteins is greatly hampered by difficulties. Aquaporin, a biological membrane protein of great medical and industrial importance, are integral proteins that form pores in cell membranes and facilitate the transport of water between cells. This protein is the membrane protein that allows the passage of water molecules and rejects all other solutes. In the literature, there is a number of researches on the discovery of aquaporin from different cell membranes.While aquaporins, which are at the forefront of biomimetic membrane production in prokaryotic cells in the industrial area, are known for the passage of molecules that provide the vital conditions of the cell in plant cells and their necessity during some stress conditions (drought), in mammalians aquaporins are associated with many diseases, especially leukemia. The biggest difficulty encountered during the studies carried out in this direction is the obstacles in ensuring the overexpression of the integral biological membrane protein aquaporins and the optimization of their purification afterwards. Aquaporins have a similar basic structure: aquaporin monomers consist of six transmembrane helical segments and two short helical segments surrounding cytoplasmic and extracellular vestibules connected by narrow aqueous pore. Between the helices are five regions (A-E) with an asparagine-proline-alanine ("NPA motif") pattern, two of which are hydrophobic (B, E) that fold in or out of the cell membrane. Another part of the channel is the "ar/R selectivity filter", a set of amino acids that allows aquaporin to selectively allow or block the passage of different molecules. Aquaporin monomers can assemble as tetramers in membranes, with each monomer functioning independently. The primary function of most aquaporins is to transport water across cell membranes in response to osmotic gradients created by active solute transport. Aquaporins provide a 10-100-fold increase in water transfer across the cell membrane. Because of their unique properties, they need to be produced in large quantities for different purposes, including structure and activity analysis or new biomimicry materials. Different species of aquaporins may have different permeability and rejection properties for the same solute. While aquaporin may vary depending on solute concentrations, the selectivity of aquaporin also varies depending on load and size. It creates a positively charged energy barrier due to the amino acid Arginine in its structure. In this way, aquaporin prevents the passage of loaded or neutral solvents. Over the past 5 years, there has been great interest in the biology of aquaporins (AQPs) in over 500 studies published on aquaporin cloning, genetics, tissue localization, developmental and regulated expression, transgenic mouse models, and structure/function analyzes. When aquaporins, which are found in all cells from bacteria to mammals, are investigated in the literature, it is seen that mostly AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8 species are water specific. AQP3, AQP7 and AQP9 are called aquaglyceroporins and also carry glycerol and/or other small uncharged molecules. Most studies on recombinant aquaporins have so far been functional, regulatory or structural studies of aquaporins. In addition to these studies, the application of aquaporins with high water permeability and high solute rejection with biomimetic membranes for water desalination and reuse has attracted great interest in recent years. Prokaryotic aquaporins were first identified in Escherichia coli, and microorganisms such as Rhodobacter sphaeroides, Lactococcus lactis, Saccharomyces cerevisiae, Mathanothermobacter marburgensis, Photobactetrium profundum SS9 and Halomonas elongate have been used in studies to purify aquaporin protein. Also the recombinant production of aquaporin is being studied by using cell-free expression systems. Studies with prokaryotic AQPs show that bacteria help cope with osmotic, oxidative stress and nutritional fluctuations. However, there are limited studies in the literature for aquaporins produced from prokaryotic organisms and especially prokaryotes under extreme conditions. Virgibacillus sp. AGTR strain isolated from Acıgöl (Burdur, Turkey), which is a lake with high salinity has taken its place in the literature as a new halophilic bacterium strain. The genome information of this organism was obtained, and it was determined that it contains two aquaporin-encoding genes, one of which is 831 bp and the other 795 bp. Also, a new thermophilic bacterium Geobacillus thermoleovorans ARTR1 was isolated from another extreme environment, Armutlu (Yalova). The gene sequence encoding aquaporin with a length of 819 bp was determined in this organism. To analyze the stability of extremophilic aquaporins in industrial processes requiring harsh conditions (high temperature, high pH), in total, three extremophilic aquaporin genes were isolated from both halophilic Virgibacillus sp. AGTR and thermophilic Geobacillus thermoleovorans ARTR1 strains. These isolated genes were transferred to the pET28a (+) vector and produced recombinantly in the Escherichia coli C43 host cell. In this process, genes related to the PCR method with appropriate primers were amplified and ligated with a pET28a (+) vector. IPTG induction was subjected to time, temperature, and molarity trials, respectively. Then the optimal condition of 1 mM IPTG, 37 °C and 4 hours induction was selected. Under this optimal condition, gene expression was visibly achieved. Since it was observed that the protein remained in the inclusion body formed in the centrifuge after sonication, it was continued with the pellet after centrifugation and examined by SDS-PAGE in the control supernatant. After this process, the protein was obtained purely by various His-tag purification methods. Proteins expected to appear at an average size of 29 kDa were confirmed by SDS-PAGE and Western Blot Analysis. Liposomes are small artificial vesicles of spherical shape that can be formed from cholesterol and non-toxic natural phospholipids. Because of their size and hydrophobic and hydrophilic character (as well as biocompatibility), liposomes are promising systems for drug delivery. Liposomes can mimic the cell membrane. Different lipid compositions can be used to obtain the desired liposome type. DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), an example of Phosphatidylcholines (PC), was used in this study. The proteoliposome consists of liposomes containing aquaporin. For 2.5 mg/ml DOPC lipid, aquaporin with a concentration of 0.5 mg/ml was dissolved in 10 ml of PBS and 0.05M OG detergent was added to obtain a proteoliposome, and aquaporin-free liposome was obtained. Control liposome and proteoliposome were treated with 0.85M NaCl and the water transfer rate of aquaporins was measured with a steady flow light scattering spectrometer device. Here, it was determined that the water passage of liposomes with aquaporin was faster. Aquaporin-bearing proteoliposomes lose water at a much higher rate than control liposomes. This was explained as an indication that aquaporin obtained from the Geobacillus Thermoleovorans ARTR1 strain was working. The identification and characterization of novel aquaporin proteins from both halophilic and thermophilic bacteria within the scope of this project are of great importance in terms of their contribution to the literature.