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Cell-free composition for ATP regeneration and use thereof

A cell-free composition, ATP synthase technology, applied in the field of cell-free systems, which can solve the problems of low efficiency and cost

Pending Publication Date: 2022-07-08
BIOCHEMINSIGHTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, these additional pathways introduce additional complexity to the overall cell-free system, with attendant inefficiencies and expense

Method used

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  • Cell-free composition for ATP regeneration and use thereof
  • Cell-free composition for ATP regeneration and use thereof
  • Cell-free composition for ATP regeneration and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1. Preparation of lipids, polymers and hybrid liposomes

[0136] Liposomes can be prepared from soybean L-α-phosphatidylcholine (95%, Avanti Polar Lipids) dissolved in 2:1 chloroform-methanol (V / V) and stored at -20°C until use. First, 10 mg of the solubilized lipids were settled in a round-bottom glass bottle, and then the solvent was evaporated under a gentle stream of nitrogen gas. Can be used containing 20mM HEPES (pH 7.5), 2.5mMMgS0 4 , 50 mg / ml sucrose in vesicle buffer to rehydrate lipid films and resuspend at a final lipid concentration of 10 mg / ml by gentle vortexing. The suspension of multilamellar vesicles (MLV) was subjected to 7 freeze-thaw cycles (1 min in liquid nitrogen, then in a 35°C water bath until fully thawed, followed by 30 sec vortex). Finally, the suspension was extruded (21 times) through 100 nm pores (polycarbonate membrane, Whatman) to homogenize the size and number of layers of the liposomes in the suspension.

[0137] Films of hy...

Embodiment 2

[0139] Example 2. Protein co-reconstitution into liposomes, polymers and hybrid liposomes

[0140] The optimized protocol for membrane protein reconstitution is as follows. Briefly, for a theoretical protein / liposome ratio of about 1 ATP synthase and 2-10 ETC, 0.14 mM pre-formed liposomes (100 pL) can be mixed with 0.14 mM sodium cholate in the presence of 0.4% sodium cholate. mMATP synthase and 0.70 mM ETC were mixed. The reconstituted mixture can be incubated at room temperature for 30 minutes with gentle agitation, and then the detergent is removed using a prepacked size exclusion column (PD MiniTrap G-25, GE Healthcare). To determine the lower production limit of the energy regeneration system, 1 ATP synthase can be reconstituted per liposome. Higher rates of ATP synthesis are readily achieved as the number of enzymes per vesicle increases. Considering the random orientation of the reconstituted ETCs, excess enzymes can be incorporated into nanocontainers (5 per liposom...

Embodiment 3

[0145] Example 3. Determination of respiration-driven ATP production

[0146] Respiration-driven ATP production can be measured as follows.

[0147] in 480 μl of measurement buffer (20 mM Tris-P0 4 (pH 7.5), 10 μl luciferin / luciferase assay (CLSII, prepared according to manufacturer’s protocol), 2 μl ADP (8.45 mM stock, ultrapure) and 1 μl DTT (1 M stock) for 10 μl liposomes , and the baseline can be recorded. Reactions can be started by adding 1 μl of ubiquinone Q1 (10 mM stock) and ATP synthesis can be recorded. At the end of each measurement, 3 [mu]l of ATP (5 mM stock) can be added to normalize the signal against the determined amount of ATP. ATP productivity can be reported as the mean of at least 3 individual preparations (each measured in 3 replicates) and the standard deviation.

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Abstract

A method of providing electrons to an electron transport chain capable of generating a proton gradient to regenerate ATP from ADP using an electrochemical cell, in particular a membrane bioreactor. Such electron transport chains may be part of a synthetic membrane, or included within the synthetic membrane, or may be prepared by appropriately disrupting living cells. The electrons provided by the electrochemical cells are delivered to the electron transport system through suitable electron carriers, such as NADH2, FMNH2, FADH2, reducing ubiquinone, thiol or other electron carriers or biologically reducing equivalents compatible with electron transport chain components that enable ATP regeneration.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims priority to US Provisional Patent Application Serial No. 62 / 804,448, filed February 12, 2019, the disclosure of which is incorporated herein by reference in its entirety. technical field [0003] The present invention generally relates to cell-free systems for the regeneration of adenosine triphosphate (ATP), which is widely used in various biologically mediated reactions such as peptide and protein synthesis. Background technique [0004] Cell-free synthesis is now recognized as an efficient method for producing valuable commercial compounds via biological processes without the need for classical fermentation; various methods of regenerating ATP have also been described to allow cell-free systems to perform operations that require ATP, such as peptides Amino acid coupling in synthesis. ("Prokaryotic cell-free systems development for synthetic biology"; Abel C. Chiao, Richard M. Murray, Zachary ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
CPCC12P19/36C12Y106/05003C12N9/0036C12N9/001C12Y103/05001C12Y110/02002C12N9/0055C12N9/0053C12Y109/03001C12N9/96C12P19/32C12N9/14C12N11/04C12Y306/03014
Inventor W·阿米格D·多兹
Owner BIOCHEMINSIGHTS
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