Part:BBa_K1470004
Secreted Alkaline Phosphatase (SEAP)
SEAP is a hydrolyase which derives from the human placental alkaline phosphatase and is secreted into the medium by the cell.
Phosphatases
Phosphatases cleavage phosphordiester bonds and release anorganic phosphate groups. Since water is required for this type of reaction, phospatases belong to hydrolases and are divided into two large subfamilies depending on the pH-value required for their highest activity. This group of enzymes is widespread in nature and play an important role in signalling pathways, cell proliferation and osteostasis.
Acid Phospatases
Human acid phosphatases share their maximum turnover within a low pH range but are despite that fact differing in sequence length, mass, tissue or chromosom origin. The lysosomal acid phosphatase, prostatic acid phosphatase, erythrocytic acid phosphatase, macrophage acid phosphatase, and osteoclastic acid phosphatase are examples of clinical relevance. Human acid phosphatases in general are expressed at low concentrations but changes are observed throughout several diseases like prostate cancer or Gaucher's disease [1].
Alkaline Phospatases
In general human alkaline phosphatases are built up of four segments: tissue non-specific alkaline phosphatases, germ cell alkaline phosphatases, intestinal alkaline phosphatases and placental alkaline phosphatases [2]. They need a high pH-value to catalyze reactions. Structural studies in E. coli helped to decipher the reaction mechanism and to prove its conservation in mammalian alkaline phospatases. The active centrum contains two zinc and one magnesium ion [3]. Step one is the activation of a conserved serine by a zinc atom, followed by the formation of a covalent phosphoserine intermediate and the release of phosphate or its transfer to a phosphate acceptor [4]. Unlike procaryotic versions, mammallian alkaline phosphatases are uncompetitively inhibited by various L-amino acids like leucine, phenylalanine, tryptophane or homoarginie [5].
Secreted human placental alkaline phosphatase
Human placental alkaline phosphatase. The monomer I is shown in pale green, monomer II in blue, N-terminal α-helix in red, crown domain in orange, and metal binding domain in yellow [6].
The human placental alkaline phosphatase is one of three tissue specific alkaline phosphatases. It shows high sequence similarity with all remaining human alkaline phophatases [6]. It is believed that this protein plays a role in cell divison in both normal and transferred cells [7]. Due to the phosphatidylinositol-glycan-anchor, the human placental alkaline phosphatase is a membrane-bound protein. The hydrophobic moiety is added post-translationally after removing 29 amino acids from the C-Terminus. A single substitute in the previous hydrophobic domain by a charged residue prevents addition of the phosphatidylinositol-glycan-anchor and leads to the secreted human placental alkaline phosphatase (SEAP) [9].
SEAP is an ideal reporter protein and a significant tool for eucaryotic promotor studies [9] and part of many commercial kits. The big advantage is its self-excretion into the medium. It can be easily measured with para-nitrophenylphosphate as a substrate. Cleavaeging the phosphate group leads to the formation of para-nitrophenol, which can be detected with light of 405 nm wave length.
A SEAP assay was performed in a 96 well plate, as described above.
The measured SEAP units were portrayed in pseudocolors. Pink wells were set to green and yellow wells are shown as blue spots.
Regarding the reaction kinetics, the measured activity in the medium is linearly proportional to changes of inracellular protein and mRNA levels. The whole procedure takes merely about 60 minutes and is very precisely. It 's suitable to detect substances in a concentrastion range of 1 ng - 1 µg/ml.
The most accurate SEAP assays to date use chemiluminescent substrates such as 1,2-dioxetane CSPD whicg are also fast and easy to perform as well.
The results of the blue light systems were measured by SEAP expression:
Figure 4: Efficiency of the blue light system using different time intervals of illumination. To test the efficiency of the light system, SEAP expression after illumination was determined. The SEAP assay was performed 24 hours after light exposure. Cells were incubated with blue light for 1 hour, 2.5 hours and 5 hours.
More informationReferences
[1] Acid phosphatases, H Bull, P G Murray, D Thomas, A M Fraser, P N Nelson, Mol Pathol. 2002 April; 55(2): 65–72.[2] Structural evidence of functional divergence in human alkaline phosphatases, Marie-Hélène Le Du, Jose Luis Millan, J Biol Chem. 2002 December 20; 277(51): 49808–49814.
[3] Structural studies of human placental alkaline phosphatase in complex with functional ligands., Paola Llinas, Enrico A. Stura, André Ménez, Zoltan Kiss, Torgny Stigbrand, José Luis Millán, Marie Hélène Le Du, J Mol Biol. 2005 July 15; 350(3): 441–451.
[4] 3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity., L. Chen, D. Neidhart, W. M. Kohlbrenner, W. Mandecki, S. Bell, J. Sowadski, C. Abad-Zapatero, Protein Eng. 1992 October; 5(7): 605–610.
[5] Organ-specific inhibition of human alkaline phosphatase isoenzymes of liver, bone, intestine and placenta; L-phenylalanine, L-tryptophan and L homoarginine., W. H. Fishman, H. G. Sie, Enzymologia. 1971 September 30; 41(3): 141–167. [6] Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity., M. H. Le Du, T. Stigbrand, M. J. Taussig, A. Menez, E. A. Stura, J Biol Chem. 2001 March 23; 276(12): 9158–9165.
[7] Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression., A. A. Dabare, A. M. Nouri, H. Cannell, T. Moss, A. K. Nigam, R. T. Oliver, Urol Int. 1999; 63(3): 168–174.
[8] Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol- glycan-anchored protein to a secreted protein., J Cell Biol. 1992 February 1; 116(3): 799–807.
[9] Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells., J. Berger, J. Hauber, R. Hauber, R. Geiger, B. R. Cullen, Gene. 1988 June 15; 66(1): 1–10.
Contribution: iGEM TU-Eindhoven 2022
SEAP is the most commonly used secreted serum reporter to monitor gene expression in eukaryotic cells.1 Therefore, the iGEM TU-Eindhoven team extended the documentation of SEAP with an overview of the advantages of using SEAP as a reporter protein. In addition, this page is extended with data of a colorimetric SEAP assay, which is supplemented with both an elaborated description and a MATLAB script to calculate the SEAP activity.
Usage and Biology
SEAP is a useful reporter protein to investigate the amount of transcriptional activity of enhancer/promoter elements.2 Because SEAP is naturally secreted by mammalian cells, its use as a genetic reporter provides several advantages: 1) the SEAP assay does not require the preparation of cell lysates, 2) during the SEAP activity measurement the transfected cells remain unharmed, 3) the gene expression kinetics can be studied easily, as the medium of the same cultures can repeatedly be collected, 4) the culture medium does not contain significant background from endogenous alkaline phosphate activity, 5) the usage of multi-well plates allows for easy automation of sample collection and assays. Because of these advantages, SEAP reporter systems are appropriate for high-throughput applications.2,3
Characterization – Colorimetric SEAP assay
iGEM TU-Eindhoven team 2022further characterized SEAP by performing a colorimetric SEAP assay. SEAP was cloned into pLS13; a mammalian reporter plasmid for STAT3-induced SEAP expression (Ostat3-PhCMVmin-SEAP-pA).4 Together with pLS15; a vector for mammalian STAT3 expression (PhCMV-STAT3-pA)4 and pLeo619-PSV40; a mammalian expression vector to recombinantly express the EpoRm-IL-6RBm-pA fused to the anti-RR120 camelid heavy chain antibody VHHA52 (GenBank accession no. MG437012)5, pLS13 was transfected into HEK293T cells that were seeded on a 24-well plate. Each well contained 0.24·106 cells. After transfection, the HEK293T cells were treated with the ligand azo dye RR120, following 48 hours of incubation at 37°C and 5% CO2. Subsequently, the medium was aspirated from the cells, to perform the SEAP assay. Figure 1 shows an overview of the transfected HEK293T cells.
Figure 1 | Design of the engineered cells. The binding of the ligand RR120 to the anti-RR120 VHH domain results in the activation of the GEMS receptor. Through the JAK/STAT pathway, gene expression and secretion of SEAP will be induced.
To perform the colorimetric SEAP assay, the Secreted Alkaline Phosphatase Reporter (SEAPorterTM) Assay Kit (Novus Biologicals, catalog# NBP2-25285) was used. In this assay, the colorless alkaline phosphatase substrate p-nitrophenyl phosphate (pNPP) is added to the collected medium containing secreted SEAP. As a consequence, SEAP catalyzes the hydrolysis of pNPP (Figure 2).
Figure 2 | Hydrolysis reaction of pNPP catalyzed by SEAP. Adapted from: Dorobanłu et al.6
This reaction results in the yellow-colored p-nitrophenol (pNP). Following the production of this product, the absorbance was measured every half a minute for one hour at a wavelength of 405 nm by the Tecan Spark plate reader. Hereof, the catalytic activity of SEAP was calculated (Figure 3). The figure demonstrates significant SEAP activity after treating the cells with 100 and 300 ng/mL RR120. This indicates that activation of the GEMS receptor was obtained after treating the HEK293T cells with these concentrations of RR120.
Figure 3 | SEAP activity calculated from the absorbance at 405 nm measured with the plate reader. HEK293T cells transfected with plasmids encoding for the anti-RR120 GEMS receptor, the STAT transcription factor, and the SEAP reporter protein, were treated with different concentrations of the ligand RR120. After 48 hours of incubation, in which the cells produced SEAP, the cell culture medium was aspirated and a colorimetric SEAP assay was performed. The absorbance at 405 nm was measured every half a minute for 1 hour. Subsequently, from this measurement, the SEAP activity was calculated. n=3 biological replicates were used.
Calculation of SEAP activity
The absorbance at 405 nm was measured in absorbance units (AU). To determine the SEAP activity, a calibration curve was made by measuring samples containing a known concentration of pNP (Figure 4). This calibration curve is specifically made for the plate reader that has been used to measure the absorbance. Importantly, using a different plate reader requires a calibration curve made for that specific plate reader to allow accurate calculations. As a result, certain absorbance values have been measured for known concentrations of pNP.
Figure 4 | The calibration curve obtained by measuring the absorbance from a titration of known concentrations of pNP. A straight line was fitted through the measured absorbance values. This line was described by the formula: Absorbance [AU] = a · pNP[mM] + b. This calibration curve is specifically obtained for the plate reader that was used for the measurement of the colorimetric SEAP assay.
This calibration curve was used to convert the measured absorbance of the colorimetric SEAP assay into concentrations of the product pNP present in the cell culture medium samples. Per sample, this conversion was done for all measurements that were obtained during one hour of measuring. This resulted in a graph containing data points that equal the concentrations of pNP measured over time. Through these data points, a straight line was fitted. The slope of this line determined the conversion of pNPP to pNP by SEAP per minute. As a known concentration of pNPP was added to the sample, the SEAP activity, expressed in enzyme units per liter (U/L), which equals μmol/min/liter, could be calculated from this slope. One enzyme unit equals the enzyme activity by converting 1 μmol of pNPP by SEAP, per minute.7
MATLAB script
This MATLAB script was used to calculate the SEAP activity.
References
1 Tannous BA, Teng J. Secreted blood reporters: Insights and applications. Biotechnol Adv. 2011;29(6):997. doi:10.1016/J.BIOTECHADV.2011.08.021
2 Kain SR, Ganguly S. Overview of Genetic Reporter Systems. Curr Protoc Mol Biol. 2004;68(1). doi:10.1002/0471142727.MB0906S36
3 Yang TT, Sinai P, Kitts PA, Kain SR. Quantification of Gene Expression with a Secreted Alkaline Phosphatase Reporter System. https://doi.org/102144/97236pf01. 2018;23(6):1110-1114. doi:10.2144/97236PF01
4 Schukur L, Geering B, Charpin-El Hamri G, Fussenegger M. Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis. Sci Transl Med. 2015;7(318). doi:10.1126/SCITRANSLMED.AAC4964/SUPPL_FILE/7-318RA201_SM.PDF
5 Expression vector pLeo619, complete sequence - Nucleotide - NCBI. Accessed September 8, 2022. https://www.ncbi.nlm.nih.gov/nuccore/MG437012
6 Dorobanłu I, Radu M. SYNTHESIS AND CHARACTERIZATION OF THE ENZYMATIC MARKER NANDROLONE-3-CARBOXYMETHYLOXIME-ALKALINE PHOSPHATASE TO BE USED IN ELISA TECHNIQUE FOR ASSAYS OF NANDROLONE FROM BIOLOGICAL SAMPLES. 2007;17(1):45-54.
7 McDonald AG, Tipton KF. Parameter Reliability and Understanding Enzyme Function. Molecules. 2022;27(1). doi:10.3390/MOLECULES27010263
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 209
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 520
Illegal NgoMIV site found at 1489 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 850
Illegal BsaI.rc site found at 1339
| None |