Expression and characterization of human lactoferrin with tandem zinc finger protein in Chlamydomonas reinhardtii

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.