This technology uses extracts produced from yeast transformed with a new anti-UV DNA construct to block ultraviolet (UV) radiation.
The anti-UV DNA construct was prepared using synthetic biology and/or conventional molecular biology. Different genetic sequences, including repair proteins — such as heat shock protein (HSP), energy metabolic proteins, and other up-regulating proteins — and other synthetic gene sequences were used.
The anti-UV DNA construct was assembled with complementary synthetic sequences such as ribosomal switch, ribosomal binding site (RBS), iron promoter, and yellow fluorescent protein reporter (YFRP). The map of the assembled anti-UV DNA construct is shown in Figure 1. This was cloned into bake yeast cells. The transformed yeast cells were grown, and the extract of the yeast cells was isolated as a powder (Figure 2) and evaluated for anti-UV activity on a human skin culture.
The efficacy of the technology was evaluated using in-vitro and in-vivo tests. These tests were performed by molecular and toxicological analyses. The yeast cells were subjected to protein expression using two-dimensional DIGE analysis based on fluorescence detection with a laser beam as a photonic source. The extract was also subjected to biochemical analysis using HPLC, mass spectrometry, and electrospray techniques. The safety of the extract was determined by LD50 in-vivo tests. The ability of the extract to provide UV protection was demonstrated by treating human skin cell cultures with the extract.
Yeast cells transformed with the anti-UV DNA construct expressed higher amounts of proteins for environmental protection related to heat or cell repair — such as heat shock protein (HSP) and energy metabolism proteins (alcohol dehydroge-nase and hexokinase) — that are involved in UV mediation mechanisms. Conversely, non-transformed cells (control) showed lower protein expression (Figure 3).
The application of this technology was also demonstrated when skin cell (fibroblast) cultures treated with extract produced from the transformed yeast cells were protected against UV radiation. Skin cells (fibroblast) treated with the extract exhibited a higher rate (>80 %) of survival after being exposed to UV radiation (254 nm and 365 nm) for different periods of time (up to 24 hours) when compared to non-treated skin cells (control), which showed a much lower survival rate (<30%) against UV radiation. The extract also exhibited a good toxicological safety range and good solubility in dimethyl sulfoxide (DMSO), commonly used as a carrier and/or for solubility test.
This work was done by Raul Cuero Ph.D. of BioCapital Holdings, and David S. McKay, Ph.D. of NASA’s Johnson Space Center. For more information, visit here.