engBratanov, DmitryGordeliy, ValentinBüldt, GeorgExpression, purification, and crystallization of bacteriorhodopsin and its derivativesinfo:eu-repo/classification/ddc/550Geowissenschaftenbacteriorhodopsinmembrane proteinexpressioncrystallizationEnergy production in a living cells is among the most important questions in biology and for the modern technology. Bacteriorhodopsin (bR) being a simplest tool to produce electric potential across membrane have received a lot of attention. It became one of the model membrane proteins, also because of its relative abundance in nature and relative ease of purification from natural source, H. salinarum. Unique properties of bR photocycle make it useful and promising in a wide variety of technical applications, thus giving rise to growing need of this protein. Despite the availability of the atomic structures there are still controversies in mechanism of proton pumping by bR. Homologous production of bR and its mutants in halobacteria is laborious, time-, and resource-consuming, therefore facile and robust E. coli expression system would be of wide interest. Recently several structures of GPCRs were obtained using GPCR-lysozyme fusion protein where lysozyme served as a crystallization tag. We suppose that bR would be a good model for investigation of versatility of lysozyme as crystallization tags. As in meso grown crystals of bR are prone to twinning crystallization of bR-lysozyme fusion protein could provide twinning-free crystals allowing to clarify the details of bR photocycle. In this work, it was suggested that the low yield of bR expression in E. coli can be attributed to the poor insertion of the protein into membrane. We have introduced protein complementary approach that may allow to localize the problem in membrane protein expression using finite number of steps. It is based on constructing of chimeric proteins between a protein of interest and complementary homologous protein expressed with high yield. Applying this approach we showed that the substitution of first ten amino acids of bR for the corresponding eight amino acids from SRII increase the expression yield of bR more than 50-fold. The reason for high yield of the chimera could be the positively charged Arg7 on the N-terminus of bR that deviates from “positive inside” rule and absent in the chimera. We expressed bR mutants R7Q and R7E where this positive charge was substituted for neutral and negative charges, respectively. Although the yields of the mutants were higher than of wild type gene, they were still considerably lower than yield of chimera. Thus, the positive charge on the N-terminus of bR is not the reason of its poor expression in E. coli. A putative stem structure 5'-end of bR mRNA was proposed to be another reason of low bR expression in E. coli. The expression yield of optimized wild type bR gene was on the same level as yield of the chimera. Therefore, the low yield of bacteriorhodopsin native gene in E. coli was attributed to the unfavorable mRNA structure of native gene close to the ribosome binding site. When purified under non-denaturing conditions, the protein have retained its functionality. The yield of functional homogenious protein was 2.4±1.3 mg per liter of culture what is sufficient for a large-scale crystallization and industrial use. Using this approach we produced as well functional V49A, D85N, and D96N mutants of bR in short time with yields of 0.3, 3.8, and 8.8 mg per liter of culture, respectively. We suppose that increased yield of D85N and D96N mutants can be explained by better incorporation of positively charged C-helix of bR into E. coli membrane. The second goal, crystallization of the bR-lysozyme fusion protein, demands a high yield expression system and effective crystallization approaches. Here, bR can serve as a reference. High resolution structures show that bR trimers are surrounded by the belt of native lipid. Despite multiple protocols of E. coli expression, 3D crystallization of this protein was not reported. Since expressed in E. coli bR-lysozyme fusion protein would not have H. salinarum lipids bound and purification of this protein is based on the application of DDM instead of usual OG, careful investigation of the influence of lipid/detergent environment on in meso crystallization is important. To study the influence of detergent on the in meso crystallization we set large-scale crystallization trials with homologously expressed bR in mixtures of detergents. The crystals of different size (up to 300 μm) were obtained. Three crystals grown in mixtures of detergents radiation gave at synchrotron a diffraction up to 1.45 Å. The full datasets were collected and three structures of bR were solved. We have not observed detergent molecules on the electron densities corresponding to the structures. These experiments showed that detergent molecules do not participate in the formation of the crystal lattice of bR. Moreover, there is no need to exchange the detergent from DDM used for purification to OG that is generally used for in meso crystallization of homologously expressed bR. Then, using the protein expressed E. coli the crystals of wild type bR and D85N and D96N bR mutants were grown. Crystals were tested under synchrotron radiation and gave a diffraction up to 2.5Å resolution. The first 3D crystals of bR expressed in E. coli demonstrate that expression of bR and its mutants in E. coli is suitable for scientific and industrial applications. In addition, the successful crystallization of protein isolated from E. coli demonstrated that H. salinarum lipids are not strictly required for grow of well ordered bR crystals. Using the optimized bR gene we have expressed bR-lysozyme fusion proteins in E. coli with yield up to 0.9 mg of functional protein per liter of culture. This protein was purified under non-denaturing conditions to homogeneity and in meso crystallization trials are ongoing.Aachen : Publikationsserver der RWTH Aachen University 109 S. : Ill., graph. Darst. (2016). = Aachen, Techn. Hochsch., Diss., 2014info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionPublikationsserver der RWTH Aachen University2016info:eu-repo/semantics/openAccessDEhttps://publications.rwth-aachen.de/record/480785https://publications.rwth-aachen.de/search?p=id:%22RWTH-2015-03875%22StudentsStudent Financial Aid ProvidersTeachersResearchersinfo:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-rwth-2015-038758