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Membrane proteins are mainly reflected by biofilm function that involves many important biological processes, a gene encoding a membrane protein of the total of 30% of protein-coding genes, in-depth study of membrane proteins to clarify the basis of the laws of life activities is extremely important . However, most of the natural membrane protein content is very low in the cell membrane, purification is extremely difficult, serious constraints on the membrane structure and function of proteins, membrane proteins with innovative drug development targets, as well as the production of membrane protein antibody. Therefore, in vitro recombinant expression of membrane proteins become an important research methods and tools, but it also has many difficulties, especially in expressing membrane protein (integral membrane proteins, IMPs). Expression of heterologous or homologous expression of transmembrane protein often shows low, no functional activity, or the formation of insoluble inclusion bodies, or the host toxicity. In addition, expression of membrane proteins into the host cell can correctly is difficult to predict and control. Therefore, many researchers are committed to efficient expression of membrane proteins in vitro, purification and crystallization of exploration and development, in order to promote membrane protein structure and function studies. Glucose is the major metabolic cellular substrates and energy substances. Facilitated glucose transporter (Facilitates glucose transporters, GLUTs) uptake and transport of glucose, is a key regulator of cellular metabolism. Therefore, the study of the structure and function of GLUTs, to better understand the process of their glucose metabolism and mechanism of action of the key, but depends on a large number of purified, functional activity of GLUT proteins. Since eukaryotic membrane proteins, especially multiple transmembrane protein expression and purification difficult, so far, there is no high-level expression in vitro GLUTs reported. In this study, the use of molecular biology techniques, access to the human GLUT family GLUT1, GLUT2 and GLUT3 three family members of the full-length gene was constructed pESP-GLUTs expression vector, to achieve a GST-GLUT1, GST-GLUT2, GST-GLUT3 fusion protein In S. pombe High expression. PESP-2 on the expression vector promoter nmt1 strictly regulated by thiamine concentration; GST fusion protein by affinity chromatography step purified. 1 liter of yeast culture fluid collected about 3 grams wet weight yeast, up to about 300 micrograms obtained was purified fusion protein. GLUT1, GLUT2 and GLUT3 contain twelve transmembrane domains, suggesting that eukaryotic transmembrane proteins can be effective in Schizosaccharomyces pombe expression and purification. Meanwhile, the experiment also with GLUT1 example, analyzes the different culture conditions, different growth conditions yeast RNA expression levels of protein expression, protein production, and host-related gene expression. Experimental results show that: 1. Usually selected Schizosaccharomyces pombe optimal growth conditions (28-30oC, pH 5.5) is not membrane-bound membrane proteins (i.e. with the host cell membrane protein on the part of) the best expression conditions; 2 total film obtained can not be used as protein expression of membrane-bound expression of membrane proteins instructions are not necessarily linked between them; 3 in the same culture conditions, the type of yeast growth state, the membrane protein RNA Membrane protein expression levels showed no significant relationship; 4 different culture conditions, the membrane-bound part of the differential expression of membrane proteins involved in the secretory pathway of the host membrane proteins differentially expressed genes related. In addition, the present study, GST-pulldown technology, verify GLUT1 with a 3 - GAPDH (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) interactions. SDS-PAGE and WB results strongly suggest that the expression of GLUT1 interacts with GAPDH in vitro, indicating that in fission yeast heterologous expression of GLUT1 has some biological activity. Access to high membrane protein antibody has been a problem. Currently, most of the membrane protein antibodies are obtained using the peptide fragments immunized animals, does not apply to all experimental study. In this study, GLUT1 protein purified immune Balb / c mice produced polyclonal antibodies. By SDS-PAGE and WB experiments prove that the antibody is homogeneous, highly specific polyclonal anti-GLUT1 mice. We believe that this antibody against GLUT1 in subsequent studies play an important role. Finally, we conducted a GLUT protein family evolutionary analysis. We from a variety of protozoa, fungi, invertebrates and vertebrates genomes isolated GLUT family members, their phylogenetic tree was constructed to analyze the evolution of the GLUT family history. The results showed that the most significant GLUT family differentiation occurs in invertebrates and vertebrates differentiation, the fish and before the divergence of terrestrial vertebrates. Nonsynonymous mutation rate and the ratio of synonymous mutation rate (Ka / Ks) indicates that mammalian GLUT very similar, functional area is very conservative, but fish and mammals GLUT quite different from the other evidence in the GLUT terrestrial vertebrates like fish and the occurrence of a greater differentiation. In addition, we also separately analyzed the evolutionary tree of GLUT1 by bioinformatics tools for the three-dimensional structure of GLUT1 conserved regions were analyzed, the results showed that GLUT1 and non-conserved regions of conserved transmembrane region is in each alternating . GLUT1 protein sequences in their family has 58 most conservative amino acid sites, which contains a number of experiments have proven the importance of GLUT1 functional sites. In GLUT1, in addition to proven QLS and QLG these two important motif, we also found another new one very conservative motif (NAP), the motif may be functionally similar QLS is the glucose transporter GLUT1 necessary.
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