Use of plant-derived recombinant growth factors in skin care
A growth factor and skin technology, applied in the application field of plant-derived recombinant growth factors in skin care, can solve problems such as interference with the immune system, instability, and carrying
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Embodiment 1
[0068] Example 1: Partially purified transgenic plant extracts containing recombinant growth factors and dehydrin Fetch
[0069] Transgenic plant extracts were prepared by milling harvested transgenic barley seeds containing the recombinant growth factor mouse stem cell factor (mSCF) in a grinder to obtain a fine powder (flour). Extraction buffer (50 mM potassium phosphate, pH 7.0) was added to the milled barley flour at a volume / weight ratio of extraction buffer to milled flour of 5 / 1. The resulting solution was stirred at 4°C for 60 minutes. The solids were separated from the liquid extract by centrifugation at 8300 rpm or above for 15 minutes in a refrigerated centrifuge (Heraeus Primo R) and the supernatant was decanted into a new vial. The growth factor content of the extracts was analyzed by SDS-PAGE and Western blotting using specific antibodies. In this experiment, the content of mSCF was about 0.1% of the protein content of the unpurified extract.
[0070] The ...
Embodiment 2
[0071] Example 2: Extraction of recombinant growth factor mSCF purified from transgenic barley seed extracts purification
[0072] For further isolation of the growth factor in purified form: the IMAC eluate from Example 1 was applied to an ion exchange column Sepharose FF after buffer shift using gel filtration and carried out by increasing the NaCl content of the elution buffer Gradually eluted to separate the protein in the extract. In this way, growth factors can be successfully separated from dehydrin. Bands are blurred due to glycosylation of mSCF. as figure 1 In this way, it was shown that growth factors could be purified to a high purity of >95% (lane 6), resulting in isolated and purified mSCF isolated and purified from transgenic plant extracts.
[0073] figure 1 Transgenic plant extracts and purification and isolation of murine stem cell factor (mSCF) from transgenic plant extracts are shown.
[0074] A) Coomassie blue stained SDS-PAGE gel staining the extr...
Embodiment 3
[0078] Example 3: Glycosylation of recombinant growth factors expressed in plants
[0079]Human gamma-interferon was expressed in barley and the extraction of total protein was carried out under reducing conditions in the presence of 170 mM NaCl, 1% 2-mercaptoethanol, 10 mM Tris-HCl pH 8.0 and 1% polyvinylpyrrolidone (MW 360.000). After the samples had been ground, 5 ml of extraction buffer was added to the extraction bottle. Extracts were clarified by centrifugation at 4000 rpm for 10 minutes at 4°C, followed by 10-fold centrifugal concentration in Ultrafree-4 concentrators using a 5 kDa molecular weight cut-off (UFV4BCC00-Millipore Corp. Bedford, MA, USA). Add 100 μl of the clarified and concentrated extract sample to 100 μl of 2x sample buffer and place the mixture in a boiling water bath for 5 min. After cooling, 10 μl of samples were loaded onto a 12% polyacrylamide gel and separated by SDS-PAGE. The results of the comparative examples were illustrated by Western blot...
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