Part:BBa_K1216005

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

Figure 3. Cell lysate from E.Coli overexpressing PhoA-His after reacting with 4-MU-phosphate.

Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-phosphate. The picture was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

Figure 4. Enzymatic reaction of PhoA with 4-MU-phosphate.

To prove that the enzyme retained functionality with an additional His-tag they conducted fluorometric assays to obtain K_m values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc ETHZ system 2013]). After another 4-5 h of growth, cells were harvested and lysed, the cell free extract (CFX) used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.

Figure 5. Michaelis-Menten-Kinetics of PhoA-His cell lysate from E.Coli overexpressing PhoA-His: plots velocity versus substrate concentration (2.5 μL, 5 μL, 10 μL, 30 μL, 60 μL, 120 μL, 240 μL) in 20 mM Tris buffer of pH 8. A kinetic value for K_m obtained by fitting the raw data to standard the Michaelis Menten equation; K_m = 72.0 ± 7.3 μM. All assays were carried out in triplicates, results are presented as means.

The experimental procedure was as following:

Prepare buffers
- Lysis buffer: 10 mg/ml Lysozyme, 20 mM Tris buffer, pH 8
- Reaction buffer: 20 mM Tris buffer, pH 8
- NOTE: For other enzymes than the ones we tested (Aes,GusA,NagZ,PhoA) you might need different buffers
Cell culture
- Inoculate bacteria in 20 mL of LB with antibiotics
- Let grow at 37°C shaking(200 rpm) to an OD₆₀₀ of 0.6
- Induce enzyme expression (100nM AHL in our case)
- Let grow at 37°C shaking(200 rpm) for 4-5h
Cell lysis
- Transfer to 50 mL Falcon™ tube
- Spin down at 4°C for 5 min with 4 rcf
- Resuspend in lysis buffer, 1 μL/mg pellet
- Transfer to eppendorf tubes
- Incubate at room temperature for 10 min at 220 rpm
- Spin down at 4°C for 10 min with max. speed
- Transfer the supernatant to new tubes, discard pellets
- Cell free extract can be stored at -20°C or continue processing
Dilution
- The following values were provided by Johannes Haerle
  - Aes: Dilute CFX 1:100 in reaction buffer
  - GusA: Dilute CFX 1:100 in reaction buffer
  - NagZ: Use pure
  - PhoA: Dilute CFX 1:10 in reaction buffer
Hydrolysis reaction
- Perform this measurement in a 96 well plate or similar
- Perform 3 replicates for each substrate concentration
- Present 41.6 μL reaction buffer in each well
- Add 8 μL diluted CFX (the further dilution ocurring here is intended)
- Add 30.4 μL of corresponding substrate
- Detection of fluorescence in suitable plate reader (λEx 365 nm, ΛEm 445 nm)

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.