Expression and Purification of Functional Human Mu Opioid Receptor from E.coli

Ma, Yanbin; Kubicek, Jan; Labahn, Jörg
February 2013
PLoS ONE;Feb2013, Vol. 8 Issue 2, p1
Academic Journal
N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3–0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a KD of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.


Related Articles

  • Cloning, expression, and isolation from Escherichia coli of human protein SURF-6 translationally fused to glutathione-S-transferase. Kordyukova, M.; Zatsepina, O.; Polzikov, M. // Applied Biochemistry & Microbiology;Mar2011, Vol. 47 Issue 2, p123 

    DNA of human gene Surf-6 ( hSutf-6) was amplified and cloned into vector pGEX-2T for the expression in the bacterial system of protein hSURF-6 translationally fused to glutathione-S-transferase. The resulting vector is named as pGEX-2T-GST-hSurf-6. Superproducer of chimeric protein GST-hSURF-6...

  • Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801. Liu, Nan; Mao, Xiangzhao; Yang, Meng; Mu, Bozhong; Wei, Dongzhi // World Journal of Microbiology & Biotechnology;Jun2014, Vol. 30 Issue 6, p1691 

    agWH50C, a novel β-agarase gene, was cloned from Agarivorans gilvus WH0801 by degenerate PCR and nested PCR. The gene agWH50C comprized a 2,223-bp, encoding a protein of 740 amino acids. Sequencing results demonstrated that AgWH50C shared 45 % sequence identity with a well characterized...

  • Growing Gene Network by Integration of Gene Expression, Motif Sequence and Metabolic Information. Bo Geng; Xiaobo Zhou; Hung, Y. S.; Wong, Stephen // AIP Conference Proceedings;11/2/2007, Vol. 952 Issue 1, p279 

    In computational biology, gene networks are typically inferred from gene expression data alone. Incorporating multiple types of biological information makes it possible to improve gene network estimation. In this paper, we describe an approach for growing gene network from a sub-network by the...

  • The expression of nifB gene from Herbaspirillum seropedicae is dependent upon the NifA and RpoN proteins. Rego, Fabiane G. M.; Pedrosa, Fábio O; Chubatsu, Leda S.; Yates, M. Geoffrey; Wassem, Roseli; Steffens, Maria B. R.; Rigo, Liu U.; Souza, Emanuel M. // Canadian Journal of Microbiology;Dec2006, Vol. 52 Issue 12, p1199 

    The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a –24/–12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of...

  • Odorant Binding Characteristics of Three Recombinant Odorant Binding Proteins in Microplitis mediator (Hymenoptera: Braconidae). Li, Keming; Wang, Shanning; Zhang, Kang; Ren, Liyan; Ali, Abid; Zhang, Yongjun; Zhou, Jingjiang; Guo, Yuyuan // Journal of Chemical Ecology;Jun2014, Vol. 40 Issue 6, p541 

    Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. In this study, three new putative OBP genes, MmedOBP8- 10, were identified from a Microplitis...

  • Growth rate-coordinated transcriptome reorganization in bacteria. Yuki Matsumoto; Yoshie Murakami; Saburo Tsuru; Bei-Wen Ying; Tetsuya Yomo // BMC Genomics;2013, Vol. 14 Issue 1, p1 

    Background Cell growth rate reflects an organism's physiological state and largely relies on the ability of gene expression to respond to the environment. The relationship between cellular growth rate and gene expression remains unknown. Results Growth rate-coordinated changes in gene expression...

  • Patterning Bacterial Communities on Epithelial Cells. Dwidar, Mohammed; Leung, Brendan M.; Yaguchi, Toshiyuki; Takayama, Shuichi; Mitchell, Robert J. // PLoS ONE;Jun2013, Vol. 8 Issue 6, p1 

    Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive...

  • Genome-Wide Analysis of Selective Constraints on High Stability Regions of mRNA Reveals Multiple Compensatory Mutations in Escherichia coli. Mao, Yuanhui; Li, Qian; Zhang, Yinwen; Zhang, Junjie; Wei, Gehong; Tao, Shiheng // PLoS ONE;Sep2013, Vol. 8 Issue 9, p1 

    Message RNA (mRNA) carries a large number of local secondary structures, with structural stability to participate in the regulations of gene expression. A worthy question is how the local structural stability is maintained under the constraint that multiple selective pressures are imposed on...

  • Multiple Genetic Pathways for Restarting DNA Replication Forks in Escherichia coli K-12. Sandler, Steven J. // Genetics;Jun2000, Vol. 155 Issue 2, p487 

    Shows that priA2::kan causes synthetic lethality when placed in combination with either Deltarep:kan or priC303:kan in Escherichia coli. Primosome assembly proteins; Ability to restart DNA replication forks at recombinational intermediates; Application of a nonselective P1 transduction-based...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics