Expression of plant sweet protein brazzein in the milk of transgenic mice

Abstract

Sugar, the most popular sweetener, is essential in daily food. However, excessive sugar intake has been associated with several lifestyle-related diseases. Finding healthier and more economical alternatives to sugars and artificial sweeteners has received increasing attention to fulfill the growing demand. Brazzein, which comes from the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is a protein that is 2,000 times sweeter than sucrose by weight. Here we report the production of transgenic mice that carry the optimized brazzein gene driven by the goat Beta-casein promoter, which specifically directs gene expression in the mammary glands. Using western blot analysis and immunohistochemistry, we confirmed that brazzein could be efficiently expressed in mammalian milk, while retaining its sweetness. This study presents the possibility of producing plant protein-sweetened milk from large animals such as cattle and goats.

Figure 1. Schematic of pCAG–Brazzein-neo and pBC1-Br-loxP-neo-loxP.

(A) Brazzein was directionally cloned into the EcoRI and XhoI sites of the pCAG vector. (B) Brazzein was cloned into the XhoI site of the pBC1 vector. The resulting constructs are composed of the following genetic elements: globin insulator, goat Beta-casein promoter, goat Beta-casein exon 1, intron 1, and part of exon 2 (before initial goat Beta-casein codon); brazzein, bovine Beta-casein signal peptide gene, and an optimized brazzein coding region; casein E7–E9, goat Beta-casein exon 7, intron 7, exon 8, intron 8, and exon 9; casein 3′ genomic DNA, and goat Beta-casein 3′ genomic region.

Figure 2. Expression of brazzein in HEK-293 cell line.

(A) Western blot analysis of the lysate and supernatant of HEK-293 cells transfected with pCAG-Br-neo; the lysate and supernatant of HEK-293 cells transfected with pCAG-neo were used as the negative control. (B) Immunofluorescence assay of the lysate and supernatant of HEK-293 cells transfected with pCAG-neo and pCAG-Br-neo. Scale bars = 100 um.

Figure 3. Identification of transgenic mice via PCR and Southern blot analysis.

(A) PCR analysis of brazzein in mES cells. WT, non-transfected cells, ddH2O were used as templates in the negative controls; 1 to 15: genomic DNA from selected stable cell clones as templates; P = positive control of brazzein gene amplified via PCR using the pBC1-Br-loxP-neo-loxP vector as the template. (B) Screening of stable transgenic mES cell clones using G418 selection method. (C) Germline chimeric mouse and the offspring of chimeras. (D) PCR amplification of genomic DNA from transgenic mice. M is the DNA ladder; WT is the non-transgenic mouse, and P is the positive control. Numbers 1, 2 and 3 are different brazzein transgenic mice. (E) Southern blot analysis of mouse genomic DNA from the tails: Genomic DNA was isolated from wild-type and transgenic mice (1, 2, and 3), then digested with HindIII. The digested DNA were resolved on a DNA gel and blotted to a nitrocellulose member. The membrane was subjected to hybridization with the brazzein probe. NC, a non-transgenic mouse control, 1, 2 and 3, the transgenic mouse line; positive controls were done with 1, 5, or 10 copy numbers.

Figure 4. RT-PCR analysis of transgenic mouse from various tissues to determine relative transcripts levels of brazzein.

(A) RT-PCR was performed to determine the tissue specificity and expression levels of brazzein in transgenic mice. The tissues analyzed were from liver (li), heart (ht), spleen (sp), lungs (lu), kidneys (ki), pancreas (pa), uterus (ut), and mammary glands (mg). M represents Marker; -RT represents no M-MuLV Reverse Transcriptase in the reaction to rule out genomic DNA contamination. The bottom panel is the result of the RT-PCR analysis of the mouse GAPDH gene. (B) The relative transcripts of brazzein in mammary gland tissues detected by real-time PCR were much higher than in other tissues. Total mRNA was extracted from mammary tissues of mouse lines 1, 2, and 3.The results are shown as means ± S.D. ***P<0.001 compared with control.

Figure 5. Detection of brazzein in the milk of transgenic mice.

(A) Expression of brazzein in the mammary glands of transgenic mice under western blot analysis. Skim milk samples (1 uL each) were separated by SDS-PAGE and immunoblotted with anti-brazzein antibodies; WT, non-transgenic milk; 1, 2, and 3, milk from transgenic mice; 3-F2a to 3-F2e represent the milk produced by the F2 generation of mouse line (B) Immunofluorescent histochemical stains of brazzein in two sections of the mammary gland tissues. Non-transgenic mammary gland tissues were used as the negative control. Scale bars = 50 um. (C) Transgenic mouse mammary gland tissues. Scale bars = 50 um. (D) Hematoxylin and eosin staining of transgenic mammary gland tissues. Scale bars = 50 um. (E) Brazzein concentrations in the milk of different mice. Control, the milk of a non-transgenic mouse; 1, 2, 3, 3-F2a, 3-F2b, 3-F2c, 3-F2d, and 3-F2e are milk samples from the different transgenic mice.

Figure 6. Sweetness test of recombinant brazzein.

(A) Sweetness was scored from 1 to 4 as follows: (0) not sweet, (0.5) uncertain if sweetness was tasted, (1.0) faintly sweet, (2.0) sweet, (3.0) very sweet and (4.0) extremely sweet. Results of psychophysical experiments with brazzein are shown; data were averaged for the 14 volunteers. Error bars represent SD. Column patterns indicate different levels of sweetness compared with the control (non-transgenic milk); the others are the sweetness of milk from the different transgenic mice. The results are shown as means ± S.D. ***P<0.001 compared with control.