Difference between revisions of "Part:BBa K1216005"

(→‎Characterization)
(→‎Characterization)
Line 25: Line 25:
  
 
===Characterization===
 
===Characterization===
 +
'''Western Blot'''
 
[[File:phoahis.png|500px|left]]
 
[[File:phoahis.png|500px|left]]
−
<p align="justify">The ''phoA'' and ''phoA with His-Tag'' were overexpressed in the ''Escherichia coli''BL21 DE3 strain, induced by 5 &mu;M IPTG (iso-propy-&beta;-D-1-thiogalactopyranoside) and finally  harvested in order to obtain the cell lysate by lysozyme lysis. 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-6X His tag® antibody from mouse and a second reporter goat anti mouse antibody with a IRDye 680RD. 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).</p>
+
<p align="justify">The ''phoA'' and ''phoA with His-Tag'' were overexpressed in the ''Escherichia coli'' BL21 DE3 strain, induced by 5 &mu;M IPTG (iso-propy-&beta;-D-1-thiogalactopyranoside) and finally  harvested in order to obtain the cell lysate by lysozyme lysis. 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-6X His tag® antibody from mouse and a second reporter goat anti mouse antibody with a IRDye 680RD. 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).</p>
 
<br clear="all"/>
 
<br clear="all"/>
 +
 +
'''Fluorescent and colorimetric substrate'''
 +
[[File:phoahis_fl.png|500px|left]]
  
 
===References===
 
===References===

Revision as of 15:22, 4 October 2013

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

Western Blot

Phoahis.png

The phoA and phoA with His-Tag were overexpressed in the Escherichia coli BL21 DE3 strain, induced by 5 μM IPTG (iso-propy-β-D-1-thiogalactopyranoside) and finally harvested in order to obtain the cell lysate by lysozyme lysis. 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-6X His tag® antibody from mouse and a second reporter goat anti mouse antibody with a IRDye 680RD. 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).


Fluorescent and colorimetric substrate

Phoahis fl.png

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.