The recombinant pig Aminopeptidase N (ANPEP) can be produced in multiple expression systems, including yeast, in vivo biotinylation in E. coli, baculovirus, and mammalian cells. The gene encoding the partial pig ANPEP protein is cloned into an expression vector, which is transformed into the expression system. If needed, any tag could be added to the target gene. The cells are cultured under conditions that promote protein expression. After sufficient growth is achieved, the cells are lysed to release the recombinant ANPEP protein, which is subjected to affinity chromatography purification. The purity of the protein is confirmed using SDS-PAGE, reaching up to 85%.
ANPEP, also known as CD13, is a type II membrane glycoprotein belonging to the family of membrane-bound metalloproteases. It is expressed in various cells, including porcine enterocytes [1]. ANPEP has been identified as an endocytotic F4 receptor in pigs, enhancing the intestinal mucosal immune response [2]. This transmembrane metalloprotease is highly expressed in immune cells, activated endothelial cells, certain epithelial cells, and fibroblasts at sites of inflammation [3]. ANPEP plays a crucial role in protein and peptide degradation, cell invasion, migration, and angiogenesis [4]. It cleaves N-terminal neutral amino acids from peptides and proteins [5]. ANPEP is overexpressed in several tumor cell malignancies, where it mediates the intracellular hydrolysis of compounds like Mel-flufen [6].
ANPEP has been studied in viral infections, with some coronaviruses, including transmissible gastroenteritis virus (TGEV), utilizing it as a receptor for cell entry [7]. Research has demonstrated that ANPEP has the potential as a biomarker and therapeutic target in diseases like gallbladder carcinoma and atherosclerosis [4][8].
Furthermore, ANPEP has been linked to various physiological processes, such as impaired angiogenesis in mice lacking ANPEP [9] and delayed mammary gland development in mice deficient in ANPEP [10]. The enzyme's multifaceted functions extend to its involvement in cancer dissemination, making it a target for anti-cancer agents [11].
References:
[1] V. Melkebeek, K. Rasschaert, P. Bellot, K. Tilleman, H. Favoreel, D. Deforceet al., Targeting aminopeptidase n, a newly identified receptor for f4ac fimbriae, enhances the intestinal mucosal immune response, Mucosal Immunology, vol. 5, no. 6, p. 635-645, 2012. https://doi.org/10.1038/mi.2012.37
[2] T. Goetstouwers, M. Poucke, V. Nguyen, V. Melkebeek, A. Coddens, D. Deforceet al., F4-related mutation and expression analysis of the aminopeptidase n gene in pigs1, Journal of Animal Science, vol. 92, no. 5, p. 1866-1873, 2014. https://doi.org/10.2527/jas.2013-7307
[3] M. Ghosh and L. Shapiro, Cd13 regulation of membrane recycling: implications for cancer dissemination, Molecular & Cellular Oncology, vol. 6, no. 6, p. e1648024, 2019. https://doi.org/10.1080/23723556.2019.1648024
[4] R. Priya, V. Jain, J. Akhtar, G. Chauhan, P. Sakhuja, S. Goyalet al., Plasma-derived candidate biomarkers for detection of gallbladder carcinoma, Scientific Reports, vol. 11, no. 1, 2021. https://doi.org/10.1038/s41598-021-02923-7
[5] A. Kido, S. Krueger, C. Haeckel, & A. Roessner, Untitled, Clinical & Experimental Metastasis, vol. 20, no. 7, p. 585-592, 2003. https://doi.org/10.1023/a:1027383729767
[6] D. Chauhan, A. Ray, K. Viktorsson, J. Spira, C. Paba‐Prada, N. Munshiet al., in vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells, Clinical Cancer Research, vol. 19, no. 11, p. 3019-3031, 2013. https://doi.org/10.1158/1078-0432.ccr-12-3752
[7] W. Li, R. Luo, Q. He, F. Kuppeveld, P. Rottier, & B. Bosch, Aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry, Virus Research, vol. 235, p. 6-13, 2017. https://doi.org/10.1016/j.virusres.2017.03.018
[8] Y. Li, Q. Jia, H. Cao, P. He, X. Shen, Y. Huanget al., Effect of bushen jiangzhi recipe on atherosclerosis in apoe−/- mice by regulating the expression of anpep via mmu_circrna_22187, Evidence-Based Complementary and Alternative Medicine, vol. 2021, p. 1-10, 2021. https://doi.org/10.1155/2021/4738264
[9] R. Rangel, Y. Sun, L. Guzman-Rojas, M. Ozawa, J. Sun, R. Giordanoet al., Impaired angiogenesis in aminopeptidase n-null mice, Proceedings of the National Academy of Sciences, vol. 104, no. 11, p. 4588-4593, 2007. https://doi.org/10.1073/pnas.0611653104
[10] A. Kolb, D. Sorrell, C. Lassnig, S. Lillico, A. Carlisle, C. Neilet al., Mammary gland development is delayed in mice deficient for aminopeptidase n, Transgenic Research, vol. 22, no. 2, p. 425-434, 2012. https://doi.org/10.1007/s11248-012-9654-7
[11] S. Amin, N. Adhikari, & T. Jha, Design of aminopeptidase n inhibitors as anti-cancer agents, Journal of Medicinal Chemistry, vol. 61, no. 15, p. 6468-6490, 2018. https://doi.org/10.1021/acs.jmedchem.7b00782
