Revision as of 13:29, 7 October 2022

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

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.

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.

References

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 2022 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 2022 further characterized SEAP by performing a colorimetric SEAP assay. SEAP was cloned into pLS13; a mammalian reporter plasmid for STAT3-induced SEAP expression (O_stat3-P_hCMVmin-SEAP-pA).⁴ Together with pLS15; a vector for mammalian STAT3 expression (P_hCMV-STAT3-pA)⁴ and pLeo619-P_SV40; a mammalian expression vector to recombinantly express the EpoR_m-IL-6RB_m-pA fused to the anti-RR120 camelid heavy chain antibody VHH_A52 (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.

To perform the colorimetric SEAP assay, the Secreted Alkaline Phosphatase Reporter (SEAPorter^TM) 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).

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.

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.

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

The MATLAB script that was used to calculate the SEAP activity can be found here and on the wiki page of the iGEM TU-Eindhoven team 2022.

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