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CN-121991991-A - System and method for improving membrane protein integration efficiency in artificially synthesized cells

CN121991991ACN 121991991 ACN121991991 ACN 121991991ACN-121991991-A

Abstract

The invention provides a system for improving the integration efficiency of a membrane protein in an artificially synthesized cell, which comprises a DNA template for encoding the target membrane protein, an amphipathic molecule combined with a phospholipid membrane and a3 '-untranslated region of an mRNA sequence, and an in vitro transcription and translation synthesis system, wherein the DNA template for encoding the target membrane protein comprises a locating sequence region template, the locating sequence region template is transcribed to obtain a locating sequence, the locating sequence is positioned in the 3' -untranslated region of the mRNA sequence transcribed from the DNA template for encoding the target membrane protein, and the locating sequence is specifically combined with the amphipathic molecule. The invention also provides a method for improving the integration efficiency of the membrane protein in the artificial synthetic cell, which comprises the step of preparing the artificial synthetic cell by using the system of the invention and adopting a reverse emulsification method. The system and the method can realize efficient membrane protein synthesis and membrane protein integration, and improve the stability and the functionality of the membrane protein in the artificially synthesized cells.

Inventors

  • LU YING
  • FU HANG
  • LI MING

Assignees

  • 中国科学院物理研究所

Dates

Publication Date
20260508
Application Date
20241106

Claims (10)

  1. 1. A system for improving the integration efficiency of a membrane protein in an artificially synthesized cell comprises a DNA template for encoding the target membrane protein, an amphipathic molecule combined with a phospholipid membrane and a3 '-untranslated region of an mRNA sequence, and an in vitro transcription and translation synthesis system, wherein the DNA template for encoding the target membrane protein comprises a locating sequence region template, the locating sequence region template is transcribed to obtain a locating sequence, the locating sequence is positioned in the 3' -untranslated region of the mRNA sequence transcribed from the DNA template for encoding the target membrane protein, and the locating sequence is specifically combined with the amphipathic molecule.
  2. 2. The system of claim 1, wherein the nucleotide sequence of the localization sequence region template comprises the nucleotide sequence set forth in SEQ ID No. 1; preferably, the nucleotide sequence of the localization sequence region template comprises the nucleotide sequence as shown in SEQ ID NO. 5.
  3. 3. The system according to claim 1, wherein the amphipathic molecule is cholesterol modified single stranded DNA, preferably the cholesterol modified single stranded DNA is complementarily paired with the localization sequence to form a pairing region, more preferably the pairing region is no less than 20bp in length; further preferably, the nucleotide sequence of the cholesterol modified single stranded DNA comprises the nucleotide sequence shown as SEQ ID No. 2; Most preferably, the nucleotide sequence of the cholesterol modified single stranded DNA comprises the nucleotide sequence shown as SEQ ID No. 3.
  4. 4. The system of claim 1, wherein the DNA template encoding the membrane protein of interest further comprises a coding region and a promoter in sequence upstream of the localization sequence region template, and optionally the DNA template encoding the membrane protein of interest further comprises an mRNA tracer sequence located between the localization sequence region template and the coding region.
  5. 5. The system of any one of claims 1 to 4, wherein the synthetic cell is a giant unilamellar vesicle.
  6. 6. The system of any one of claims 1 to 4, wherein the membrane protein of interest is an alpha helix-rich membrane protein; Preferably, the target membrane protein is selected from any one or more of LacY protein, emrE protein and bacteriorhodopsin protein.
  7. 7. A method for increasing the efficiency of membrane protein integration in artificially synthesized cells, comprising the steps of: (1) Preparing an emulsifying system; (2) Mixing the system for improving the integration efficiency of membrane proteins in artificially synthesized cells according to any one of claims 1 to 6 with the emulsifying system to obtain a mixture; (3) Centrifuging the mixture to prepare artificial synthetic cells; (4) Incubating the synthetic cells.
  8. 8. The method of claim 7, wherein in step (1), the emulsifying system is prepared by mixing phosphatidylcholine with mineral oil; preferably, the ratio of the weight of phosphatidylcholine to the volume of mineral oil is (0.1-2) mg: 200. Mu.L; More preferably, the ratio of the weight of phosphatidylcholine to the volume of mineral oil is 1mg:200 μl.
  9. 9. The method according to claim 7, wherein, in the step (2), the ratio of the volume of the system for improving the integration efficiency of the membrane protein in the artificially synthesized cells to the volume of the emulsifying system is (10-20) μl (100-600) μl; Preferably, the final concentrations of the DNA template encoding the membrane protein of interest and the amphipathic molecule in the mixture in the system for improving the efficiency of membrane protein integration in artificially synthesized cells are 1-10nM and 0.5-3. Mu.M, respectively, more preferably 3nM and 1. Mu.M, respectively.
  10. 10. The method according to claim 7, wherein in the step (3), the centrifugal force of the centrifugal treatment is 15000-20000g for 15-25 minutes, preferably 18000g for 20 minutes; in step (4), the incubation is at a temperature of 30-37 ℃ for a time of 2-4 hours, preferably at 37 ℃ for a time of 3 hours.

Description

System and method for improving membrane protein integration efficiency in artificially synthesized cells Technical Field The invention belongs to the field of biotechnology. In particular, the invention relates to a system and method for improving the integration efficiency of membrane proteins in artificially synthesized cells. Background Synthetic cells serve as important branches of modern synthetic biology, imitate functions and behaviors of natural cells, and show wide application potential. With the continuous progress in the fields of molecular biology and synthetic biology, artificial synthetic cell technology has become the leading edge of bioscience research. Scientists have been able to reconstruct vital basic features in synthetic cells, such as the processes of replication of genetic material, synthesis of proteins, and cell division. Cells in nature are clearly defined by cell membranes, and various membrane proteins distributed on the cell membranes play a decisive role in the realization of cell functions, involving various aspects of signal transduction, substance exchange, energy metabolism, and maintenance of membrane structure. In order to make synthetic cells better mimic these basic biological functions and to enhance their compatibility and efficacy in complex biological systems, ensuring proper synthesis and localization of membrane proteins is also an essential key. However, in synthetic cells, the synthesis and localization of membrane proteins is a significant challenge. Traditional cell-free expression systems have difficulty in effectively mimicking the membrane protein synthesis and localization processes in natural cells. Membrane proteins containing large amounts of hydrophobic amino acids are often accompanied by misfolding and aggregation during synthesis, resulting in loss of function. Therefore, development of a novel synthetic system and method for artificial synthetic cells to improve the synthetic efficiency of the artificial synthetic cells, the efficiency and stability of the integration into cell membranes is a technical problem to be solved at present. Disclosure of Invention Membrane proteins play a critical role in cellular operations, and they play a key role in signal transduction, mass transport, energy generation, membrane structure construction, and the like. These proteins occupy 20% to 30% of the open reading frame of the genome. In the field of synthetic biology, the importance of membrane proteins is self-evident in that they enable synthetic cells to mimic basic biological functions, thereby enhancing their fusion and function in biological systems. However, in synthetic cells, the synthesis and localization of membrane proteins by cell-free systems is also faced with a number of challenges. Because of their hydrophobic nature, membrane proteins are prone to misfolding and deleterious aggregation in water. Based on the existing thermodynamic knowledge, embedding hydrophobic amino acids in random coil form into membranes requires overcoming a huge thermodynamic energy barrier. The lipid head of the lipid bilayer interface contributes to the formation of alpha-helices in the peptide chain, thereby lowering the energy barrier for membrane integration. Previous research results show that the expression level of membrane proteins in the membrane can be effectively increased by reducing the size of synthetic cells, mimicking the effects of chaperones, and using specialized ribosomes. Researchers have long noted that mRNA is not uniformly distributed in cells independent of ribosomes. In eukaryotes, mRNA encoding a membrane protein tends to be located concomitantly with the Endoplasmic Reticulum (ER), and in the prokaryotic field, certain mRNA, by virtue of its coding region sequence and structural features, is capable of interacting with the SecYEG translocase. Based on these findings, the present inventors have conducted extensive research work to creatively develop a strategy based on modifying the untranslated region (UTR) of mRNA for efficient synthesis and localization of membrane proteins in synthetic cells. The core of the present application is the introduction of a non-coding sequence located in the 3' UTR, which is referred to herein as "AnchorTail". And, cholesterol modified single stranded DN a (ssDNA) is introduced into an artificial cell, which is complementary to AnchorTail sequences, thereby achieving spontaneous base complementary pairing. Cholesterol modified single-stranded DNA is able to anchor to the cell membrane, and thus mRNA can be effectively recruited to the vicinity of the cell membrane by this pairing mechanism. The direct co-translation folding of the membrane protein on the cell membrane bilayer is promoted, and the synthesis efficiency and the positioning accuracy of the membrane protein are greatly improved. Accordingly, in view of the deficiencies of the prior art, it is an object of the present invention to provide a system