Initially, the gene fragment encoding the rat ApoE protein (19-312aa) is cloned into an expression vector, which is then introduced into a suitable expression system, including yeast cells, in vivo biotinylation in E.coli, baculovirus, or mammalian cells. Following successful introduction, the host cells are cultivated under optimized conditions to promote protein expression. After sufficient growth, the cells are harvested, and the recombinant ApoE protein is extracted and purified using affinity chromatography. The purity of this recombinant rat ApoE protein is greater than 85% as accessed by SDS-PAGE.
Apolipoprotein E (ApoE) is involved in lipid metabolism and is particularly significant in the context of neurobiology and cardiovascular health. In rats, ApoE is primarily synthesized in the liver, although it is also produced in other tissues such as the brain, adipocytes, and macrophages [1][2]. ApoE plays a vital role in the transport and metabolism of lipoproteins, facilitating the clearance of triglyceride-rich lipoproteins from the bloodstream [1][2].
Research has shown that ApoE is involved in various physiological processes, including the regulation of food intake and energy balance. Studies have indicated that ApoE expression in the hypothalamus is influenced by feeding patterns and hormonal signals, such as leptin [3][4]. The rhythmic expression of ApoE in response to food availability suggests its role in appetite regulation and energy homeostasis [3][4]. Moreover, the interaction between ApoE and corticosterone levels highlights its involvement in the neuroendocrine response to feeding [3].
In addition to its metabolic functions, ApoE's role extends to cardiovascular health, where it is involved in reverse cholesterol transport and the modulation of inflammatory responses [8][9]. The presence of ApoE in high-density lipoproteins (HDL) contributes to its antiatherogenic properties, which are crucial for maintaining cardiovascular health [8][9]. Studies have shown that ApoE deficiency in animal models leads to increased atherosclerosis and dyslipidemia, further emphasizing its protective role against cardiovascular diseases [10][11].
ApoE is also known to interact with amyloid beta peptides, which are associated with Alzheimer's disease, and plays a role in modulating neuroinflammation and neuronal survival [5][6]. The neuroprotective effects of ApoE are particularly pronounced in its isoforms, with ApoE3 being more effective than ApoE4 in regulating calcium homeostasis and protecting neurons from excitotoxicity [7].
References:
[1] Y. Li and L. Liu, Apolipoprotein e synthesized by adipocyte and apolipoprotein e carried on lipoproteins modulate adipocyte triglyceride content, Lipids in Health and Disease, vol. 13, no. 1, 2014. https://doi.org/10.1186/1476-511x-13-136
[2] P. Hauser, V. Narayanaswami, & R. Ryan, Apolipoprotein e: from lipid transport to neurobiology, Progress in Lipid Research, vol. 50, no. 1, p. 62-74, 2011. https://doi.org/10.1016/j.plipres.2010.09.001
[3] L. Shen, K. Carey, D. Wang, S. Woods, & M. Liu, Food-entrained rhythmic expression of apolipoprotein e expression in the hypothalamus of rats, Brain Research, vol. 1273, p. 66-71, 2009. https://doi.org/10.1016/j.brainres.2009.04.004
[4] L. Shen, P. Tso, D. Wang, S. Woods, W. Davidson, R. Sakai, et al., Up-regulation of apolipoprotein e by leptin in the hypothalamus of mice and rats, Physiology & Behavior, vol. 98, no. 1-2, p. 223-228, 2009. https://doi.org/10.1016/j.physbeh.2009.05.013
[5] Y. Zhao, J. Li, Q. Tang, J. Gao, C. Chen, L. Jing, et al., Apolipoprotein e mimetic peptide protects against diffuse brain injury, Neural Regeneration Research, vol. 9, no. 5, p. 463, 2014. https://doi.org/10.4103/1673-5374.130060
[6] M. Sadowski, J. Pankiewicz, H. Scholtzova, J. Ripellino, Y. Li, S. Schmidt, et al., A synthetic peptide blocking the apolipoprotein e/β-amyloid binding mitigates β-amyloid toxicity and fibril formation in vitro and reduces β-amyloid plaques in transgenic mice, American Journal of Pathology, vol. 165, no. 3, p. 937-948, 2004. https://doi.org/10.1016/s0002-9440(10)63355-x
[7] I. Veinbergs, A. Everson, Y. Sagara, & E. Masliah, Neurotoxic effects of apolipoprotein e4 are mediated via dysregulation of calcium homeostasis, Journal of Neuroscience Research, vol. 67, no. 3, p. 379-387, 2002. https://doi.org/10.1002/jnr.10138
[8] A. Valle, E. Silvestri, M. Moreno, J. Oliver, P. Roca, & F. Goglia, Combined effect of gender and caloric restriction on liver proteomic expression profile, Journal of Proteome Research, vol. 7, no. 7, p. 2872-2881, 2008. https://doi.org/10.1021/pr800086t
[9] N. Hyka, J. Dayer, C. Modoux, T. Kohno, C. Edwards, P. Roux‐Lombard, et al., Apolipoprotein a-i inhibits the production of interleukin-1β and tumor necrosis factor-α by blocking contact-mediated activation of monocytes by t lymphocytes, Blood, vol. 97, no. 8, p. 2381-2389, 2001. https://doi.org/10.1182/blood.v97.8.2381
[10] A. Cornelissen, S. Simsekyilmaz, E. Liehn, M. Rusu, N. Schaaps, M. Afify, et al., Apolipoprotein e deficient rats generated via zinc-finger nucleases exhibit pronounced in-stent restenosis, Scientific Reports, vol. 9, no. 1, 2019. https://doi.org/10.1038/s41598-019-54541-z
[11] T. Tomofuji, D. Ekuni, T. Azuma, K. Irie, Y. Endo, K. Kasuyama, et al., Involvement of toll-like receptor 2 and 4 in association between dyslipidemia and osteoclast differentiation in apolipoprotein e deficient rat periodontium, Lipids in Health and Disease, vol. 12, no. 1, 2013. https://doi.org/10.1186/1476-511x-12-1
