Alkaline purification of spider silk proteins
A spidroin protein and alkaline technology, which is applied in the field of alkaline purification of spidroin protein, can solve the problems of poor hand feeling, low fiber yield, low fiber toughness, etc.
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Embodiment 1
[0207] Example 1: Purification of 18B using single-step basic conditions
[0208] Use high pH solutions to solubilize recombinant proteins without destroying the host cells that secrete them. Test pH buffer concentration and incubation time for Argiope bruennichi expressed in P. Pastoris with a C-terminal 3x FLAG tag (SEQ ID NO: 40) Solubility of recombinant spidroin protein of the MaSp2 block ("18B", SEQ ID NO: 38). The FLAG tag was attached to the C-terminus of the 18B peptide sequence with a glycine residue (G) linker.
[0209] Specifically, cell culture fermentation broth was inoculated with Pichia pastoris expressing the 18B recombinant protein and incubated to allow expression of the 18B protein. Centrifuge the culture to harvest the cells and resuspend the cell pellet in distilled water at a ratio of 1:1 (equal parts cell pellet and water) or 1:3 (one part cell pellet and two parts water). Adjust the pH of the cell pellet suspension to a final pH of 11.8-11.9 with 2-...
Embodiment 2
[0212]Example 2: Further Purification of Isolated Silk Polypeptides
[0213] To further purify the 18B spider protein, the 18B sample isolated from the alkaline extraction above was subjected to ultrafiltration and tangential flow filtration using a 750k MW filter and 8 diavolumes of water. Samples containing unfiltered protein, ultrafiltered protein, and protein after 1, 3, 6, and 8 dialysis volumes of water were evaluated by SEC as previously described. The SEC% area of 18B monomer, medium molecular weight impurity, low molecular weight impurity and high molecular weight impurity in each sample is in Figure 5 shown in . The unfiltered protein sample ("Unconditioned Feed") is shown on the leftmost bar and the ultrafiltered protein sample ("Unconditioned UFR") is shown on the second bar from the left. ), samples with 1, 3, 6 or 8 dialysis volumes are shown in the center left, center right, second from right and rightmost bars respectively ( Figure 5 ). An increase in t...
Embodiment 3
[0214] Example 3: 18B purification using two-step alkaline extraction
[0215] To increase the recovery of 18B protein in cells, a two-step extraction process was also performed. Using 2M NaOH as the first alkaline extraction step, the pH of the whole cell broth of P. pastoris cells expressing 18B was adjusted to pH 11.8 and incubated for 30-60 minutes. A control sample of whole cell broth of P. pastoris cells expressing 18B was incubated with 5MGdSCN for about 15 minutes to solubilize and extract the 18B protein. Cells were pelleted and supernatant collected. As a second extraction step, the remaining pellet from the first alkaline extraction step was re-extracted by adding water at pH 11.8 in a pellet:water ratio of 1:1, 1:2 or 1:3. The supernatants of the first and second alkaline extractions containing the recombinant 18B protein were collected. Supernatants were lyophilized to concentrate 18B protein and samples were evaluated by SEC as previously described in Example ...
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