Method of use of stabilized plant-derived growth factor in skin care
A growth factor and skin care technology, applied in the field of cosmetic and dermatological compositions, can solve the problems of protein glycosylation and instability
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Embodiment 1
[0080] Embodiment 1: Preparation of the transgenic plant extract containing recombinant EGF
[0081] Transgenic plant extracts are prepared by grinding harvested transgenic barley seeds containing the growth factor EGF in a mill to obtain a fine powder (flour). Extraction buffer (50 mM potassium phosphate, pH 7.0) was added to the ground barley flour at a volume / weight ratio of extraction buffer to ground flour of 5 / 1. The resulting solution was stirred at 4°C for 60 minutes. Centrifuge at 8300 rpm or above for 15 min in a refrigerated centrifuge (Heraeus Primo R) to separate the solids from the liquid extract by centrifugal force and decant the supernatant into clean vials. Extracts were analyzed for growth factor content by SDS-PAGE and Western blotting using EGF-specific antibodies. In this experiment, the EGF content was about 0.01% of the protein content of the extract. The results are shown in figure 1 middle.
[0082] figure 1 (A) shows a portion of the gel stai...
Embodiment 2
[0083] Example 2: Partially purified transgenes containing the growth factors VEGF and dehydrin Plant extracts
[0084] The VEGF-containing transgenic barley seed extract prepared according to Example 1 was further processed by adding to the extract an IMAC chromatography resin that effectively binds VEGF. The mixture of extract and resin was stirred at +4°C for 60 minutes in 50 mM potassium phosphate, pH 7.0, 0.5 M NaCl, 50 mM imidazole. The IMAC resin was separated from the liquid by centrifugation at 5000 xg for 15 minutes. The liquid phase was decanted off, the resin was resuspended in wash buffer (50 mM potassium phosphate, 0.5 M NaCl, 50 mM imidazole; pH 7.0) and centrifuged, and the liquid phase was decanted from the resin. Washing was repeated three times. Resuspend the resin in elution buffer containing imidazole (50 mM potassium phosphate, 0.5 M NaCl, 500 mM imidazole; pH 7.0) to elute VEGF from the resin and decant the supernatant from the resin after centrifu...
Embodiment 3
[0086] Example 3: Extraction of partially purified transgenic plants containing hordeglobulin and IGF-1 thing
[0087] Transgenic barley seeds were extracted according to Example 1 in extraction buffer II (50 mM potassium phosphate, pH 7.0, 200 mM NaCl). The extract was bound to and eluted from IMAC resin as explained in Example 2, and the eluate was subsequently desalted and buffer changed to 100 mM KPi at pH 6.8. The extract was then further processed by adding the partially purified extract to an ion exchange chromatography resin under conditions effective to bind IGF-1, where the matrix used was SP-Sepharose (GE Healthcare). The corresponding fractions were analyzed by SDS-PAGE and silver staining and western blotting using specific anti-IGF-1 antibodies. The partially purified plant extract has an IGF-1 content of 60%.
[0088] image 3 , a partially purified transgenic plant extract containing barley globulin and IGF-1. A) Silver-stained SDS-PAGE gel. Lane numbe...
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