Self-assembled polymeric vesicular structures with functional molecules
A composition, a technology of vesicles, applied in the field of preparing selectively permeable membranes, membranes or modules comprising selectively permeable membranes, selectively permeable water membranes, water extraction, and preparation of said structures, capable of solving problems not shown
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Embodiment 1
[0065] Example 1. Dissolving PS-PAA directly in phosphate buffer at pH=7.2 in the presence of LDAO to form self-assembled vesicles
[0066] Materials and methods
[0067] Polystyrene-block-poly(acrylic acid), DDMAT-terminated (MW8000Da) (PS-PAA3000:5000, PDI3 ) 2 (CH 2 CHC 6 h 5 ) m (CH 2 CHCOOH) n SCSSC 12 h 25 ), where m=28 and n=70, and were used as received without any other purification. By adding 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 and 0.24g KH 2 PO 4 Dissolve in 800 mL MiliQ purified H 2 In O, the pH was adjusted to 7.2 with HCL and the volume was made up to 1 L to prepare a 10 mM phosphate solution (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl). N,N-Dimethyldodecylamine N-oxide BioXtra (lauryldimethylamine N-oxide) (99% purity), LDAO was purchased from Sigma Aldrich.
[0068] PS-PAA incorporated into AqpZ vesicles was prepared by LDAO-mediated direct lysis method. For this, 200 mg of PS-PAA powder was mixed with 0.5 mL of 5% LDAO stock solution and 195 mL of P...
Embodiment 2
[0072] Example 2. Purification of Universal Aquaporin and Preparation of Aquaporin Stock Solution
[0073] Recombinant Production of Aquaporin Z
[0074] Aquaporins All types and variants of aquaporins, including aquaporins, can be used to prepare membranes and compositions according to the invention, see the method described in WO2010 / 146365. Representative examples include the spinach aquaporin SoPIP2;l protein and the bacterial aquaporin-Z from E. coli.
[0075] Functional aquaporin-Z is overproduced in bacterial cultures of E. coli strain BL21(DE3) as a His-tagged protein with a tobacco etch virus cleavage site. The fusion protein has 264 amino acids and has a Mw of 27234 Da. Genomic DNA from E. coli DH5 was used as a source for amplifying the AqpZ gene. The AqpZ gene was amplified using gene-specific primers and a tobacco etch virus cleavage site (TEV) was added; ENLYFQSN is located at the N-terminus of AqpZ. The amplified AqpZ was digested with enzymes NdeI and BamHI...
Embodiment 3
[0083] Example 3. Preparation of PS-PAA vesicles with AqpZ incorporation using LDAO as surfactant
[0084] Materials and methods
[0085] Polystyrene-block-poly(acrylic acid), DDMAT-terminated (MW 8000 Da) (PS-PAA as in Example 1) was purchased from Sigma Aldrich and used as received without any other purification. By adding 8g NaCl, 0.2g KCl, 1.44gNa 2 HPO 4 and 0.24g KH 2 PO 4Dissolve in 800 mL MilliQ purified H 2 In O, adjust the pH to 7.2 with HCL and make up the volume to 1 L to prepare a 10 mM phosphate solution (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl). N,N-Dimethyldodecylamine N-oxide BioXtra (lauryldimethylamine N-oxide) (99% purity), LDAO was purchased from SigmaAldrich. ). 5 mg / mL AqpZ purified stock solution in 2% LDAO, see general preparation in Example 2 above.
[0086] PS-PAA incorporated into AqpZ vesicles was prepared by LDAO-mediated direct lysis method. For this, 200 mg of PS-PAA powder was mixed with 0.5 mL of 5% LDAO stock solution and 194.9 mL of P...
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