The Secretory Expression Of Plant Sweet Protein In The Milk Of Mammary Gland

Sweetness is the innate favorite taste of human beings. Sugar, the most popularsweetener, is essential in daily food. However, excessive sugar intake has beenassociated with several lifestyle-related diseases including caries (dental cavities),obesity, diabetes mellitus, and many more undesirable effects. Finding healthier andmore economical alternatives to sugars has received increasing attention to fulfill thegrowing demand. Sweet Protein is a kind of protein with high sweetness, low caloriesand high thermostability. So far, many sweet proteins including Brazzein, Curculin,Mabinlin, Miraculin, Monellin, Pentadin and Thaumatin have been identified. Amongthem, Brazzein, which comes from the pulp of the edible fruit of the African plantPentadiplandra brazzeana Baill, is a protein that is2,000times sweeter than sucroseby weight and has an in-diminishable sweetness profile after incubation at80℃forup to4hrs. Howerver, Brazzein can be grown only in tropical area, and the process ofpurifying Brazzein is very complicated thus limiting the production scale. Recently,the main efforts have be turned to seek producing sweet protein by geneengineering. Mammary gland bioreactor is a good factory to produce proteins with thehigh bioactivities and no toxicity. Hence, in this report, we attempt to explore thefeasibility by using mammary gland bioreactor to produce plant sweet protein.Previously, the functional expression of Δ12fatty acid desaturase (FAD2) fromspinach plants has been achieved in transgenic pigs, indicating that a large quantity ofplant protein being expressed in mammalian animal cells.To test whether the plantgene can be expressed in mammalian cells, we designed the construction with thefollowing strategy. In order to express brazzein and thaumatin well in animalmammary glands, the gene sequence was optimized by codon usage. To allowsecretion of brazzein into the milk, we used the goat secretion signal β-casein.We constructed the brazzein and thaumatin expressing vectors,pCAG-Brazzein-neo and pCAG-Thaumatin with CAG promoter for universal expression in all the tissues. Two vectors (pBC1-Brazzein and pBC1-thaumatin)which can be specifically expressed in mammary glad also were constructed. Theuniversal expression vectors were tansfected into in HEK293cells and and the sweetprotein expression have beed confirmed by both SDS-PAGE and Westernblot. To testif the mammary glad-specific expression vectors can be expressed in vivo, we directlyinjected the DNA into the rabbit mammary gland duct and the expression wasdecerned by SDS-PAGE and WB.To examine the expression of a plant protein inmammals, we generated three chimeric mouse line specifically expressing the plantprotein in the mammary gland. We transfected the vector into mouse embryo stem(mES) cell line and produced germline chimera mice. Expression of brazzein in themilk was confirmed by Western blot and immunohistochemisty. The milk oftransgenic mice is tasted sweet. These results demonstrate that a plant gene for asweet protein is able to be expressed in mammals, opening up the possibility ofproducing plant-protein-sweetened milk from those large animals as cattle and goats.