The process of producing recombinant human 3-ketoacyl-CoA thiolase, mitochondrial (ACAA2) in yeast involves co-cloning the gene of interest (17-397aa of human ACAA2) into an expression vector with an N-terminal 6xHis-tag gene and transforming it into yeast cells. The yeast cells are cultured under conditions that induce protein expression. Once adequate growth is achieved, the cells are lysed to release the recombinant ACAA2 protein. Purification is carried out using affinity chromatography. The purity of the recombinant ACAA2 protein is confirmed using SDS-PAGE, exceeding 90%.
Human ACAA2 is an enzyme is an enzyme crucial for fatty acid metabolism. ACAA2 is primarily involved in mitochondrial fatty acid elongation and degradation by catalyzing the final step of the β-oxidation pathway [1][2][3]. It is essential for converting long-chain fatty acids into acyl-CoAs, the active form utilized in cellular lipid synthesis and degradation through beta-oxidation [4]. As a liver enzyme, ACAA2 contributes to acetyl-CoA generation in mitochondria, crucial for tricarboxylic acid (TCA) cycle activity [1].
ACAA2 also exerts different roles in different contexts. For example, ACAA2 acts as a tumor suppressor in renal cell carcinoma, linked to increased immune infiltration and elevated PD-1 expression in CD8+ T cells [5]. Studies associate ACAA2 with regulating fatty acid metabolism, particularly polyunsaturated fatty acids (PUFAs), affecting lipid concentrations and cardiovascular risk factors [6][4]. Furthermore, ACAA2 is linked to protection against acetaminophen-induced hepatotoxicity through transcriptional upregulation by Egr1 [7].
References:
[1] S. Kang, D. Ren, G. Xiao, K. Daris, L. Buck, A. Enyenihiet al., Cell line profiling to improve monoclonal antibody production, Biotechnology and Bioengineering, vol. 111, no. 4, p. 748-760, 2013. https://doi.org/10.1002/bit.25141
[2] Y. Zhang, Y. Wang, X. Wang, Y. Ji, S. Cheng, M. Wanget al., Acetyl‐coenzyme a acyltransferase 2 promote the differentiation of sheep precursor adipocytes into adipocytes, Journal of Cellular Biochemistry, vol. 120, no. 5, p. 8021-8031, 2018. https://doi.org/10.1002/jcb.28080
[3] D. Miltiadou, A. Hager-Theodorides, S. Symeou, C. Constantinou, A. Psifidi, G. Banoset al., Variants in the 3′ untranslated region of the ovine acetyl-coenzyme a acyltransferase 2 gene are associated with dairy traits and exhibit differential allelic expression, Journal of Dairy Science, vol. 100, no. 8, p. 6285-6297, 2017. https://doi.org/10.3168/jds.2016-12326
[4] M. Yang, K. Liu, P. Chen, H. Zhu, J. Wang, & J. Huang, Bromodomain-containing protein 4 (brd4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis, Cell Death and Disease, vol. 13, no. 10, 2022. https://doi.org/10.1038/s41419-022-05344-0
[5] L. Le, Z. Chao, W. Un, J. Xiao, Y. Ge, Y. Wanget al., Metabolic classifications of renal cell carcinoma reveal intrinsic connections with clinical and immune characteristics, Journal of Translational Medicine, vol. 21, no. 1, 2023. https://doi.org/10.1186/s12967-023-03978-y
[6] C. Wu, H. Song, X. Fu, S. Li, & T. Jiang, Transcriptomic analysis of glioma based on idh status identifies acaa2 as a prognostic factor in lower grade glioma, Biomed Research International, vol. 2020, p. 1-8, 2020. https://doi.org/10.1155/2020/1086792
[7] X. Lei, Q. Xu, C. Li, B. Niu, Y. Ming, J. Liet al., Egr1 confers protection against acetaminophen‑induced hepatotoxicity via transcriptional upregulating of acaa2, International Journal of Biological Sciences, vol. 18, no. 9, p. 3800-3817, 2022. https://doi.org/10.7150/ijbs.71781
