Novel protein transduction domain and the usage thereof
A technology of hydrophobic interaction and serial number, applied in peptide/protein components, animal/human proteins, cosmetic preparations, etc., can solve the problems of less PTD and immune response, and achieve the effect of high yield
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Embodiment 1
[0064] [Example 1. Preparation of cell and skin-penetrating GFP protein expression vector]
[0065] In order to easily introduce macromolecular proteins or peptides into cells and skin, a recombinant protein expression vector was prepared by fusing the peptide with cell and skin penetration ability and the desired delivery protein (cargo).
[0066] In this embodiment, in order to simply analyze the transfer ability, that is, how much of such a fusion protein can be transferred to cells and skin, Green Fluorescence Protein (Green Fluorescence Protein, hereinafter referred to as for GFP).
[0067] The currently published PTD, namely TAT or PEP1, was fused to GFP, and compared with the newly developed STeP (sequence number 1) in the present invention, the intracellular and skin delivery ability was compared.
[0068] (1) Preparation of pGFP vector
[0069]A DNA fragment corresponding to the base sequence of GFP was subcloned into XhoI and BamHI positions of plasmid pET15b to pr...
Embodiment 2
[0078] [Example 2. Expression of GFP recombinant protein]
[0079] In this example, the recombinant vector prepared from the above example expresses the corresponding protein. The four expression vectors were transformed into E.coli Rosetta-gami 2(DE3) cells respectively. Transformed bacterial cells were cultured at 37°C using 1,000 ml of LB medium containing ampicillin until OD 600 When the value reached 0.6, 0.5 mM IPTG was introduced at 22° C., and further cultured for 24 hours, thereby inducing the expression of GFP protein.
[0080] The cultured cells before and after the induced expression of the target protein by adding IPTG were obtained, and after SDS-PAGE electrophoresis, the protein was stained with Coomassie brilliant blue (Coomassie brilliant blue) to observe and confirm the expression of each GFP protein.
[0081] The result is, as Figure 5 It is shown that GFP expresses a protein of 29.35 kDa, TAT-GFP expresses a protein of 30.54 kDa, PEP1-GFP expresses a pr...
Embodiment 3
[0082] [Example 3. Purification of GFP recombinant protein]
[0083] In this example, after the cell disruption step was performed on the proteins expressed in the above examples, only the GFP protein was isolated and purified using a His tag affinity column. Regarding the purification method of the four GFP proteins, purification was performed by the same method as described below.
[0084] Transformed cells with pET15-GFP, pTAT-GFP, pPEP1-GFP, pSTeP-GFP in 1X Native buffer (50mM NaPO 4 , 0.5M NaCl) in 0.5M NaCl) after being pulverized by ultrasonic equipment, it was centrifuged to separate into inclusion body and cytoplasm (supernatant).
[0085] All 4 GFP proteins have His 6 Tagging, so using this feature, the supernatants separated by centrifugation after the cells are broken use Ni 2+ -NTA affinity column (Nitrilotriacetic acid Sepharose affinity column, GE Healthcare, USA) was used for purification.
[0086] For the equilibration of the chromatographic column and the...
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