Thermosensitive hydrogels based on spider silk proteins
A temperature-sensitive hydrogel, spider silk protein technology, applied in the field of spider silk protein materials
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Embodiment 1
[0056] In this example, the expression vector of the target protein was constructed, and the expression vector was introduced into Escherichia coli to realize expression and production.
[0057] The specific operations of this example include: using two restriction enzymes, NdeI and XhoI, to construct and produce CTD 1 、CTD 2 、CTD 3 expression vector. Afterwards, different vectors were transformed into Escherichia.coli BL21 (DE3) cells, and the strain was cultured in 4 mL LB medium containing ampicillin at 37°C until the OD600 was 1.8-2.0, and then transformed into cells containing Cultivate in 100mL LB medium containing ampicillin at 37°C; when OD600 is 3.0-4.0, transfer all of them to 800mL TB medium containing ampicillin and culture at 37°C; when OD600 is 6.0-8.0, add 1mM Bacteria were harvested after induction with IPTG16°C for 12-16 hours.
[0058]The bacterial strains, plasmids, enzymes, antibiotics and culture medium involved in the above operations of this example ...
Embodiment 2
[0064] The construction of this embodiment produces (Rep) 4 -CTD 1 , (CTD 1 ) 4 expression vector, and by the method in Example 1, expression production was carried out in Escherichia coli Escherichia.coliBL21 (DE3).
[0065] In this example, a protein solution with a concentration of 150 mg / mL was obtained. The rheological properties between 0°C and 80°C were measured with a rotational rheometer (HAAKE MARS III) at a frequency of 1 rad / s and experimental conditions of 2°C / min.
[0066] The results showed that (CTD 1 ) 2 During the heating process, a phase transition (G’>G”) occurs at ~40°C, hydrogels form, and the elastic modulus (G’) changes continuously, up to 10 5 Pa (see Figure 9 ).
Embodiment 3
[0068] In this example, the bacteria obtained in Example 1 were resuspended according to the ratio of 1 g of wet bacteria to 10 mL of BufferA; lysozyme was added at a ratio of 0.5 mg / mL and incubated at 37°C for 30 minutes; after ultrasonic disruption, centrifuged at 12,000 rpm to obtain the supernatant, After filtering through a 0.45 μm filter membrane, the sample was loaded on a column filled with Ni‐Sepharose. After washing away non-specifically bound proteins with BufferB, the target protein was eluted with BufferC. Concentrate the target protein to 130-150mg / mL with a concentration tube with a cut-off flow of 10kDa, and then replace the solvent of the target protein with 20mM phosphate buffer at pH 7.2 by means of continuous addition and centrifugation.
[0069] Run the eluate of BufferC on SDS-PAGE under reducing conditions (such as figure 1 ‐2). Arrows in the figure indicate the main bands of the corresponding recombinant proteins.
[0070] The used buffer of above-m...
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Abstract
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