The gene encoding the human intestinal-type alkaline phosphatase (ALPI) protein (20-503aa) is co-incorporated into a plasmid vector with the N-terminal 10xHis-tag gene to generate recombinant plasmid, which is then introduced into mammalian cells. The mammalian cells containing the recombinant plasmid are selected and cultured under conditions that stimulate the expression of the target gene. After expression, affinity purification is employed to isolate and purify the recombinant human ALPI protein from the cell lysate. Denaturing SDS-PAGE is utilized to resolve the resulting recombinant human ALPI protein, revealing a purity level exceeding 95%. The endotoxin content of this protein is less than 1.0 EU/ug as determined by the LAL method. It has also been validated as an active enzyme. Its specific activity is greater than 8836.463 U/mg.
The protein ALPI, also known as Intestinal Alkaline Phosphatase, is an enzyme with vital roles in the body. It's found in the lining of the intestines and helps neutralize harmful substances produced by bacteria [1] [2]. ALPI's job includes calming down inflammation by dealing with these bacterial products [1]. It's crucial for keeping a healthy balance between our bodies and the bacteria living in our guts, which is important for gut health [3]. Plus, having more ALPI seems to improve the protective function of the intestinal barrier [4].
ALPI's ability to prevent inflammation is especially important in response to the gut's bacterial community [5]. It's triggered by certain bacteria products, like LPS, and works to neutralize them, preventing overly strong reactions from the body's defense system [2].
Moreover, ALPI seems to have been part of the evolutionary process of vertebrates' defense against harmful bacterial products like LPS, showing its importance across different species [6]. Its location in the intestine, where it interacts with food and the vast community of gut bacteria, suggests it's involved in many important body processes [7].
References:
[1] E. Campbell, C. MacManus, D. Kominsky, S. Keely, L. Glover, B. Bowerset al., Resolvin e1-induced intestinal alkaline phosphatase promotes resolution of inflammation through lps detoxification, Proceedings of the National Academy of Sciences, vol. 107, no. 32, p. 14298-14303, 2010. https://doi.org/10.1073/pnas.0914730107
[2] Y. Yang, J. Millán, J. Mecsas, & K. Guillemin, Intestinal alkaline phosphatase deficiency leads to lipopolysaccharide desensitization and faster weight gain, Infection and Immunity, vol. 83, no. 1, p. 247-258, 2015. https://doi.org/10.1128/iai.02520-14
[3] M. Parlato, F. Charbit‐Henrion, J. Pan, C. Romano, R. Duclaux-Loras, M. Duet al., Human alpi deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis, Embo Molecular Medicine, vol. 10, no. 4, 2018. https://doi.org/10.15252/emmm.201708483
[4] M. Núñez-Sánchez, F. Herisson, J. Keane, N. Garcia-Gonzalez, V. Rossini, J. Pinhieroet al., Microbial bile salt hydrolase activity influences gene expression profiles and gastrointestinal maturation in infant mice, Gut Microbes, vol. 14, no. 1, 2022. https://doi.org/10.1080/19490976.2022.2149023
[5] J. Bates, J. Akerlund, E. Mittge, & K. Guillemin, Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota, Cell Host & Microbe, vol. 2, no. 6, p. 371-382, 2007. https://doi.org/10.1016/j.chom.2007.10.010
[6] Y. Yang, A. Wandler, J. Postlethwait, & K. Guillemin, Dynamic evolution of the lps-detoxifying enzyme intestinal alkaline phosphatase in zebrafish and other vertebrates, Frontiers in Immunology, vol. 3, 2012. https://doi.org/10.3389/fimmu.2012.00314
[7] C. Schäfer, K. Schröder, O. Höglinger, S. Tollabimazraehno, & M. Lornejad-Schäfer, Acetaminophen changes intestinal epithelial cell membrane properties, subsequently affecting absorption processes, Cellular Physiology and Biochemistry, vol. 32, no. 2, p. 431-447, 2013. https://doi.org/10.1159/000354449"
