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Functionalized beta-sheet peptide stabilized membrane proteins, constructs comprising same, and methods of forming and using same

A membrane protein, functionalization technology, applied in chemical instruments and methods, organic chemistry methods, animal/human proteins, etc., can solve problems such as difficulty in scale, difficulty in production, and unstable structure

Inactive Publication Date: 2017-08-18
艾伯塔大学校董
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, a Langmuir-Blodgett membrane was designed in an attempt to purify water using aquaporins in a thin monolayer, but such configurations are unstable and difficult to scale (see e.g. Sun et al., 2012)
To improve stability, the monolayer was tethered to a support substrate and polymer pads were used to dampen vibrations (see e.g. Sun et al., 2013); however, these constructs also proved to be unstable and difficult to produce

Method used

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  • Functionalized beta-sheet peptide stabilized membrane proteins, constructs comprising same, and methods of forming and using same
  • Functionalized beta-sheet peptide stabilized membrane proteins, constructs comprising same, and methods of forming and using same
  • Functionalized beta-sheet peptide stabilized membrane proteins, constructs comprising same, and methods of forming and using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1.0 - Preparation of functionalized β-sheet peptide (FBP)

[0101] The chemical synthesis of FBP on embodiment 1.1-solid-phase peptide synthesizer

[0102] Using amino acids with propargyl side chains, the inventors were able to synthesize functionalized beta-sheet peptides with alkyne functionality at the C-terminus on a solid-phase peptide synthesizer. Alkyne-FBP self-assembles into filamentous structures when diluted in phosphate-buffered saline, suggesting that alkyne-FBP retains the β-sheet formation propensity of β-sheet peptides. The synthesized alkyne-functionalized β-sheet peptide has the structure of BP1 but with a C-terminal propargylglycine, i.e., acetyl-(octyl)Gly-(octyl)Gly-Ser-Leu-Ser-Leu-Asp-( Octyl)Gly-Asp-(propargyl)Gly-NH 2 , (SEQ ID NO: 12). Self-assembly of alkyne-functionalized β-sheet peptides is shown in Figure 4A middle. Figure 4B A mass spectrum showing the correct molecular mass of high purity propargyl-BP1 is shown.

[0103]...

Embodiment 12

[0104] Example 1.2 - Confirmation of Alkyne Functionality on FBP1

[0105] In order to confirm the presence and active function of the alkyne group on the alkyne-BP1 synthesized in Example 1.1, the inventors performed a reaction to react the alkyne group on the peptide with fluorescent tetrathyrhodamine azide (Click-iT kit, LifeTechnology). Click to react. The inventors then ran the reaction products on an HPLC equipped with a fluorescence detector. As expected, additional fluorescent peaks not belonging to the kit components appeared on the elution curve (at Figure 5 Indicated by arrows in the upper panel of ; the HPLC profiles of individual kits and individual dyes are shown in Figure 5 middle and bottom panels). Therefore, the inventors could assign this peak to the dye-labeled alkyne-functionalized β-sheet peptide.

Embodiment 2

[0106] Example 2.0 - Membrane protein purification and BP:MP complex formation

[0107] The inventors have purified aquaporin (AqpZ) and verified its function. AqpZ is an aqueous channel on the periplasmic membrane of Escherichia coli. AqpZ selectively transfers water at high rates and based on an osmotic gradient across the cell membrane. As noted above, existing attempts to use aquaporins in water purification applications have not been entirely successful. However, it is believed that FBP:AqpZ complexes according to the present disclosure will overcome at least some of the disadvantages associated with previous attempts.

[0108] According to standard molecular biology procedures, the inventors have (7) Overexpression and purification of recombinant AqpZ in Escherichia coli were optimized using n-dodecyl β-D-maltoside (DDM) as a solubilizing detergent. The gene encoding AqpZ was cloned using the plasmid pTrc10HisAqpZ described in literature (7) as a template. A small...

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Abstract

Constructs having membrane proteins stabilized by functionalized beta-sheet peptides are provided. The constructs can be associated with or covalently linked to supports. The support can be a membrane. The membrane can be used to selectively move desired particles from one side of the membrane to the other while impeding passage of undesired particles through the membrane. Methods of making and using such constructs and membranes are provided.

Description

[0001] References to related applications [0002] This application claims priority to and benefit of U.S. Provisional Patent Application No. 62 / 041587, filed August 25, 2014, which is incorporated herein by reference for all purposes. technical field [0003] Some embodiments of the present invention relate to functionalized β-sheet peptides and functionalized β-sheet peptide-mediated immobilization of membrane proteins. Some embodiments of the present invention relate to membrane proteins stabilized by such functionalized beta sheet peptides and uses thereof. Some embodiments of the present invention relate to constructs comprising the protein aquaporin stabilized by such functionalized beta-sheet peptides and uses thereof. Background technique [0004] Membrane proteins (MPs) comprise one-third of the genome-encoded proteins and perform essential functions as receptors, transporters, and channels. However, the isolation and purification of membrane proteins is challeng...

Claims

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Application Information

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IPC IPC(8): C07K14/245B01D61/00C02F1/44C07K14/195C07K14/32C07K14/39C07K14/415C07K14/47C07K14/705C07K17/00C07K17/08C07K17/12C07K19/00
CPCC02F1/44C07K14/705C07K17/00C07K17/08C07K17/12C07K14/47C02F2103/08B01D71/82C02F2101/12C02F2101/16C02F2101/306C02F2101/308C02F2101/38C07B2200/11C08G75/23
Inventor 卡洛·大卫·蒙泰马尼奥杰弗里·杰曼凯尔·米诺西诺伊·亚伯拉罕许晓芬何原
Owner 艾伯塔大学校董
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