Part:BBa_K2583001

ADH1_promoter-HRH4-mcherry-ADH1_terminator

This part is for heterologous expression of human histamine receptor HRH4 with mCherry tag at its C-terminal, driven by ADH1 promoter. In our project, this part was used to test the expression strength of HRH4, and to determine whether HRH4 locates in the plasma membrane as expected.

Usage and Biology

In our project, this part is used to express human histamine receptor H4 (HRH4) with mcherry tag under the trigger of a constitutive promoter—ADH1 promoter in yeast. The function is testing the expression and the location of human histamine receptor H4 (HRH4) in yeast, it lays the foundation for the subsequent experiments. HRH4 is a G-protein coupled receptor (GPCR)from Homo sapiens. H4 receptor is primarily expressed in mast cells and eosinophils, but is also found on human neutrophils and basophils, and plays an important role in modulating eosinophil chemotaxis. H4 receptor acts in concert with H2 receptor to enhance IL-16 production in lymphocytes. In mast cells, selective H4 receptor activation increases calcium influx, degranulation, cytokine release and leukotriene production (cysteinyl and LTB4). In comparison to the H1 receptor, the H4 receptor is a relatively new therapeutic target for inflammation [3,8,39]. Selective H4 receptor antagonists are in development for potential clinical use, in particular for the treatment of Th2-dependent dermal inflammation such as atopic dermatitis, and asthma. [1]

Characterization

1.Fluorescence microscopy After transforming the CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae with plasmid pGADT7 contains part BBa_K2583001, 30℃ cultivate on △leu solid medium for one day. Select monoclonal and enlarge culture overnight, then observe the fluorescence under the microscope with 580nm exciting light. The red fluorescence shows the expression of HRH4-mcherry fusion protein. 2.Flow CytoMetry (FCM) From the result of fluorescence microscopy, the expression of HRH4-mCherry fusion proteins can be observed roughly, for quantifying the expression level of HRH4-mCherry, FCM is performed. From the result, a distinct peak value can be observed. Considering the fluorescence microscopy result that most of HRH4 are correctly located, this data does provide us a reliable probability distribution of total H4 protein expression variance between yeast individuals. Finally, the probability distribution of functional H4 receptor expression variance can refer to the total expression probability distribution we get from experiments

Result

The test of this composite part set the foundation of our project, it proved that HRH4 can be expressed in high level under the trigger of a constitutive promoter-ADH1 promoter. Besides, the HRH4 can locate in the yeasts’ membrane without adding other sequence. It can be indicated that if mcherry is removed, functional HRH4 can still be expressed and locate in the membrane. Fig1. Fluorescence microscopic result of HRH4-mcherry Fig2. Fluorescence microscopic result of DIO dying and HRH4-mcherry Fig3. Flow Cytometry result of HRH4-mCherry fusion protein expression

Reference

[1] Paul Chazot, Hiroyuki Fukui, C. Robin Ganellin, Helmut L. Haas, Stephen J. Hill, Rebecca Hills, Roberto Levi, Walter Schunack, Jean-Charles Schwartz, Nigel P. Shankley, Henk Timmerman, J. Michael Young.Histamine receptors: H4 receptor. Last modified on 04/04/2018. Accessed on 18/10/2018. IUPHAR/BPS Guide to PHARMACOLOGY, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=265. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2157
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 180
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 830
  Illegal SapI site found at 2307