Expression and Bioactivity of Recombinant Human Lysozyme in the Milk of Transgenic Mice

Abstract

Human milk lysozyme is an important protein for innate immunity, but human breast milk is a fairly poor source for commercial production of this enzyme. Research on the expression of recombinant human lysozyme (rHlys) is therefore potentially valuable to the dairy industry. In this study, 2 different kinds of transgenic mice, PBC-hLY and PBC-sighLY, were generated and used as system models to express rHlys. Six lines of PBC-hLY transgenic mice with human lysozyme genomic DNA-based constructs were generated, and a maximum expression level of rHlys approaching 0.154 mg/mL was achieved. Antibacterial activity of the whey from PBC-hLY female transgenic mice was determined by a turbidimetric assay. Results showed that antibacterial activity of the whey was strongly enhanced, and confirmed that rHlys retained full activity. For rHlys to be secreted efficiently into the milk of transgenic mice, 5 lines of mice were also generated, in which the signal peptide DNA of bovine beta-casein was substituted for that of lysozyme in PBC-hLY transgenic mice. Compared with PBC-hLY transgenic mice, both the expression levels of rHlys and the antibacterial activity of the whey were much higher in the PBC-sighLY transgenic mice. The concentration of rHlys in one of these mice amounted to 1.405 mg/mL-3 times higher than the level in human whey. The antibacterial activity of the whey was also 3 times higher than that of human whey. The rHlys from both PBC-hLY and PBC-sighLY transgenic mice had the same antibacterial activity as human milk lysozyme. The effect of the signal peptide and copy numbers of the transgene on expression of rHlys was also evaluated. This work will certainly permit a better understanding of how mammary gland bioreactor systems can be applied to produce rHlys in other mammals, such as cattle.

Production of recombinant human lysozyme in the milk of transgenic pigs

“…Recently, the production of transgenic dairy goats producing human lysozyme in their milk was reported (Maga et al 2006). Our previous studies showed that the plasmid pBC1-hLZ-GFP-Neo can express bioactive rhLZ in the milk of transgenic mice (Yu et al 2006) which warrants us to further investigate the feasibility of the strategy for producing rhLZ transgenic pigs.…”

Expression and characterization of a lysine rich protein in cow milk

“…Other studies have shown that the goat -casein promoter can efficiently direct the high expression of a foreign gene in the milk of transgenic animals, such as human serum albumin (up to 40 g/L) in bovine milk (Echelard et al, 2009) lysozyme (up to 1.45 g/L) in transgenic mice (Yu et al, 2006) and lactoferrin ( 2.5 g / L and 3.4 g / L) in two transgenic cows . The levels of recombinant protein expression reported in those studies were high, suggesting that the goat -casein gene promoter is a successful expression system.…”

Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics - recent achievements

“…On the other hand, deletion from the β-casein gene of exon regions responsible for the formation of the protease (plasmin) site in its translation product can induce an increase in β-casein emulsifying properties as well as elimination of the bitter flavour in produced cheese (Hitchin et al 1996, Laible et al 2007, Pampel et al 2008. Introduction of supplementary functional copies of genes encoding regulatory immunoproteins of the milk such as lysozyme, lactoferrin or lysostaphin can enhance the antimicrobial immunosupressive activity of such genetically-modified milk (Hyvö nen et al 2006, Yu et al 2006). …”

“…It has been found that the biochemical composition of human milk differs considerably from the composition of both the milk of cattle and of small ruminants. Human milk contains much less total protein (10 g/l) than cow's milk (33 g/l), 70% of which consists of the fraction of whey proteins including, above all, lactoferrin and lysozyme (i.e., mucopeptide glycohydrolase) (Hyvö nen et al 2006, Yu et al 2006). In contrast to bovine milk, the concentration of caseins in human milk is considerably lower.…”