Expression and characterization of human lactoferrin with tandem zinc finger protein in Chlamydomonas reinhardtii
Expression and characterization of human lactoferrin with tandem zinc finger protein in Chlamydomonas reinhardtii
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人乳铁蛋白与串联锌指蛋白在莱茵衣藻中的表达和表征
DOI：
10.1016/j.algal.2019.101635
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发表时间：
2019-11-01
影响因子：
5.1
通讯作者：
Chen, Defu
中科院分区：
文献类型：
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作者：
Pang, Xiaonan;Tong, Yuxi;Chen, Defu
乳铁蛋白（LF）是一种天然存在的铁结合糖蛋白，具有多种生物学功能。然而，其生物利用度仍然是临床应用的主要限制。在这项研究中，我们探讨了功能人乳铁蛋白（hLF）串联锌指蛋白（ZFP）重复在莱茵衣藻的表达。将密码子优化的ZFPn-hLF融合基因转化到C. reinhardtii CW-15菌株。共获得23个ZFPn-hLF表达克隆。ZFPn-hLF蛋白的表达水平范围为总可溶性蛋白的1.97%至2.32%。ZFPn-hLF蛋白的表达不影响C.莱因哈德氏菌重组ZFPn-hLF保留了对大肠杆菌和变胞克雷伯氏菌IV-3的抗菌活性，以及对DPPH和羟自由基的抗氧化活性。ZFP融合不损害hLF的稳定性，并且使得hLF能够有效内化到正常人小肠细胞中。内化的效率被认为是依赖于串联ZFP的数量，孵育时间和孵育温度。我们的结果表明，重组ZFPn-hLFs在C. Reinhardtii促进了乳铁蛋白在人肠上皮细胞中的吸收，从而提供了一种有效的策略来提高乳铁蛋白的生物利用度，以用于其临床应用。
Lactoferrin (LF) is a naturally occurring iron-binding glycoprotein with a number of biological functions. However, its bioavailability remains a major limitation for clinical applications. In this study, we explored the expression of functional human lactoferrin (hLF) containing tandem zinc finger protein (ZFP) repeats in Chlamydomonas reinhardtii. Codon-optimized ZFPn-hLF fusion genes, where n donates 0, 1, 2, or 3 repeats of ZFP, were transformed into C. reinhardtii CW-15 strain. A total of 23 ZFPn-hLF-expressing clones were obtained. The expression levels of the ZFPn-hLF proteins ranged from 1.97% to 2.32% of the total soluble protein. The expression of ZFPn-hLF proteins did not compromise the proliferation of C. reinhardtii. The recombinant ZFPn-hLFs retained the antibacterial activities toward Escherichia coli and Klebsiella variicola IV-3, and also the antioxidant activities toward DPPH and hydroxyl free radicals. ZFP fusion did not compromise the stability of hLF and enabled efficient internalization of hLF into normal human small intestine cells. Efficiency of the internalization was found to be dependent on the number of tandem ZFPs, incubation time and incubation temperature. Our results indicate that recombinant ZFPn-hLFs expressed in C. reinhardtii promotes the absorption of lactoferrin in human intestinal epithelial cells, and thus provide an effective strategy to enhance the bioavailability of lactoferrin for its utilization in clinical applications.