Generator

Part:BBa_K1216005

Designed by: Parvathi Chandran   Group: iGEM13_ETH_Zurich   (2013-08-29)
Revision as of 18:45, 3 October 2013 by Sabine.oesterle (Talk | contribs) (→‎Characterization)

Alkaline phosphatase (phoA) from Citrobacter with TEV and poly-HIS tags

The alkaline phosphatase is a periplasmic homodimeric hydrolase.
3D representation of the alkaline phosphatase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ANJ RCSB]
The poly-HIS tag can be used for protein purification (IMAC)[1]. The TEV tag can then be used to have the TEV protease specifically cleave off the poly-HIS tag from the purified protein [2].

A form of this protein without TEV and poly-HIS tags can be found here.

Usage and Biology

Alkaline phosphatases are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[3]. Testing human blood for Alkaline Phosphatase levels is a routine test that can reveal different conditions[4].

Alkaline phosphatases cleave phosphate groups from organic compounds by hydrolysis while retaining stereochemistry[5]. A good explanation of the mechanism can be found [http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reactions/Section_10.3%3A_Hydrolysis_of__phosphates here].
Alkaline phosphatases, respectively their serum levels, are also related to several diseases e.g. metabolic myopathies and Paget Disease. [6]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 340
    Illegal NgoMIV site found at 787
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

PhoAcomparison.png

The phoA and phoA with His-Tag were cultivated in LB media and harvested in order to obtain the cell lysate by lyszyme treatment. This cell lysate of both cultures were analyzed next to each other in two SDS-PAGE gels, one for comassie staining (blue gel in the picture) and one for western blotting (black picture) with anti-anti His antibody gathering a red dye. In the picture we can distinguish the PhoA His (53 kDa) on the western blot as well as on the SDS-PAGE gel (see white circles)


References

  1. Loghran ST, "Purification of poly-histidine-tagged proteins.",Methods Mol Biol. 2011;681:311-35. doi: 10.1007/978-1-60761-913-0_17.[http://www.ncbi.nlm.nih.gov/pubmed/20978973]
  2. [http://homepage.univie.ac.at/nikos.pinotsis/tev_protease.html University of Vienna TEV Protease info]
  3. Molecular Cell Biology, Fifth Edition, W.H. Freeman & Co., 2004.
  4. [http://www.nlm.nih.gov/medlineplus/ency/article/003470.htm Medline Plus]
  5. [http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reactions/Section_10.3%3A_Hydrolysis_of__phosphates Section 10.3: Hydrolysis of phosphates]
  6. Adams & Victor's Principles Of Neurology, 7th edition, McGraw-Hill Professional, 2000.
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