Difference between revisions of "Part:BBa K1216005"
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===Fluorescent and colorimetric substrate=== | ===Fluorescent and colorimetric substrate=== | ||
[[File:phoahis_fl.png|200px|left]] | [[File:phoahis_fl.png|200px|left]] | ||
+ | To test the functionality of our reporter enyzme, we tested the both a flourescent and a colorimetric substrate on the liquid culture of'' E.coli'' overexpressing PhoA and PhoAHIS | ||
===Western Blot=== | ===Western Blot=== |
Revision as of 15:32, 4 October 2013
Alkaline phosphatase (phoA) from Citrobacter with TEV and poly-HIS tags
The alkaline phosphatase is a periplasmic homodimeric hydrolase. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 340
Illegal NgoMIV site found at 787 - 1000COMPATIBLE WITH RFC[1000]
Characterization
Fluorescent and colorimetric substrate
To test the functionality of our reporter enyzme, we tested the both a flourescent and a colorimetric substrate on the liquid culture of E.coli overexpressing PhoA and PhoAHIS
Western Blot
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).
References
- 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]
- [http://homepage.univie.ac.at/nikos.pinotsis/tev_protease.html University of Vienna TEV Protease info]
- Molecular Cell Biology, Fifth Edition, W.H. Freeman & Co., 2004.
- [http://www.nlm.nih.gov/medlineplus/ency/article/003470.htm Medline Plus]
- [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]
- Adams & Victor's Principles Of Neurology, 7th edition, McGraw-Hill Professional, 2000.