| It is estimated that roughly 30% of eukaryotic genes encode membrane proteins and it play key roles in a wide variety of cellular functions. The membrane proteins not only have the close relation with disease occurrence and development (about 70-80% of human disease-related protein is membrane protein); it is closely linked with the new medical exploration and research. Despite its obvious physiological importance, the structural and functional analyses of membrane proteins lag far behind than that of soluble proteins. The most important reason is that the recombinant expression and subsequent purification of IMPs is considered a major challenge, especially for eukaryotic membrane proteins. For instance, overexpression in both heterologous and endogenous hosts is often toxic and can result in the production of inactive proteins or insoluble aggregates. Proper insertion into the host membrane is also rate-limiting and remains difficult to predict or regulate. The functional and structural analysis of membrane proteins has been hampered by the lack of sufficient quantities of active proteins. Consequently, many investigators have attempted to deal with these difficulties by focusing on expression, purification and crystallization strategies.Glucose is the major energy source for mammalian cells as well as an important substrate for protein and lipid synthesis. The Facilitates glucose transporters (GLUTs) functions are uptaking glucose as a key metabolism regulator in the cellular. Therefore, studies of GLUTs structures and functions, the regulation and inhibition of their activity, their roles as drug targets and their antibody production are important for understanding the pathogenesis of disease and exploring new treatment strategies. Such studies are highly dependent on the efficient in vitro GLUT expression and, thus far, there has been no report of a system for high expression of GLUTs in vitro.In order to carry out profound studies of GLUT structures and functions, three subfamily members of the human 12-transmembrane-domain cell-surface receptors GLUT1, GLUT2 and GLUT3 were heterologously expressed in the fission yeast S. pombe by utilizing GST-GLUT fusion proteins. These fusion proteins were driven by the full-length nmt1 promoter (Pnmt1) derived from S. pombe. The transcription levels of the GST-GLUT fusion proteins were very high upon induction by removing thiamine from the media. One-step purification of the recombinant fusion proteins was achieved by GST-affinity chromatography. Approximately 300μg of highly purified fusion protein was obtained from 3g of wet cell paste (1 liter of cell culture), indicating that human membrane proteins can be efficiently expressed and purified in the fission yeast. With its available extensive genetic information and ease of genetic manipulation, the fission yeast is potentially a highly efficient host to express eukaryotic membrane proteins.In addition, we examined the reasons for successes and failures in recombinant membrane protein production (take GLUT1 as example) in fission yeast about the RNA levels of the protein under defined growth regimes and the related encoding genes expression of the host. Our data showed some conclusions:1. The most rapid growth conditions of those chosen (28-30oC, pH 5.5) were not the optimal production conditions for membrane-bound protein expression and it was crucial to harvest yeast prior to glucose exhaustion for maximum production amounts;2. The amount of membrane protein obtained in the total extract was not a good indicator of the yield of functional membrane-bound protein;3. The differences in membrane protein yields observed under different culture conditions were not reflected in corresponding changes in RNA levels of the membrane protein; and4. The differences in membrane protein yields could be related to the differential expression of the fission yeast encoding genes involved in membrane protein secretory pathway and yeast cellular physiology.Furthermore, we demonstrated the biologic activity of the expressed membrane protein GLUT1 through detecting the interaction between GLUT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using the GST fusion protein pull-down technique. The SDS-PAGE and western-blotting results verified the interactions between them in our study. It demonstrated that the expressed GLUT1 in S.pombe had, or at least partially had its biologic activity.Moreover, the purified GLUT1 allowed the production of highly specific mouse polyclonal antibodies. These antibodies have been proven specific, homogeneity, and useful for studies on western-blotting protocols. We believed that it would be applied for more other researches, such as expression analysis, interactions of proteins and functional identification and encourage the investigations of GLUT1 functions.At last, we performed the evolution analysis of GLUTs family. The identification of GLUTs from, protozoa, fungi, invertebrates, as well as vertebrates allowed us to illuminate the phylogenetic and evolutionary history of this gene family. The significant expansion of GLUT genes primarily was occured after the divergence between vertebrates and invertebrates and before the divergence between the ray-finned fish and mammals. The nonsynonymous/synonynmous substitution ratio (Ka/Ks) values showed that strong functional constraints must impose on the mammal GLUTs. However, the discrepancy between mammals and fishes was apparent, implying the existence of functional divergence between them. We analyzed GLUT1 sequences in different species, mapping the results to the three-dimensional structure of GLUT1 by using bioinformatical method. We identified the most conserved sites in the GLUT1 and found out a new motif (NAP) which is probably crucial for the selectivity of GLUTs for the transported sugars like QLS motif.
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