upscaling a metabolic network for micrometer&#45;sized vesicles

복잡한 단백질 네트워크를 위한 지질 소포 캡슐화 프로토콜

The field of bottom-up synthetic biology focuses on constructing (minimal) cells1,2 and metabolic bioreactors for biotechnological3,4 or biomedical purposes5,6,7,8. The construction of synthetic cells provides a unique platform that allows researchers to study (membrane) proteins in well-defined conditions mimicking those of native environments, enabling the discovery of emergent properties and concealed biochemical functions of proteins and reaction networks9. As an intermediate step towards an autonomously functioning synthetic cell, modules are developed that capture essential features of living cells such as metabolic energy conservation, protein and lipid synthesis, and homeostasis. Such modules not only enhance our understanding of life but also have potential applications in the fields of medicine8 and biotechnology10.
Transmembrane proteins are at the heart of virtually any metabolic network as they transport molecules in or out of the cell, signal, and respond to the quality of the environment, and play numerous biosynthetic roles. Thus, the engineering of metabolic modules in synthetic cells requires in most cases the reconstitution of integral and/or peripheral membrane proteins into a membrane bilayer composed of specific lipids and high integrity (low permeability). The handling of these membrane proteins is challenging and requires specific knowledge and experimental skills.
Several methods have been developed to reconstitute membrane proteins within phospholipid vesicles, most often with the purpose of studying the function11,12, regulation13, kinetic properties14,15, lipid dependence15,16, and/or stability17 of a specific protein. These methods involve the rapid dilution of detergent-solubilized protein into aqueous media in the presence of lipids18, the removal of detergents by incubating detergent-solubilized protein with detergent-destabilized lipid vesicles and absorption of the detergent(s) onto polystyrene beads19, or the removal of detergents by dialysis or size-exclusion chromatography20. Organic solvents have been used to form lipid vesicles, for example, via the formation of oil-water interphases21, but the majority of integral membrane proteins are inactivated when exposed to such solvents.
In our laboratory, we mostly reconstitute membrane proteins by the detergent-absorption method to form large-unilamellar vesicles (LUVs)19. This method allows the co-reconstitution of multiple membrane proteins and the encapsulation in the vesicle lumen of enzymes, metabolites, and probes22,23. The membrane protein-containing LUVs can be converted into giant-unilamellar vesicles (GUVs) with/without encapsulation of water-soluble components, using either electroformation24 or gel-assisted swelling25 and specific conditions to preserve the integrity of the membrane proteins26.
This paper presents a protocol for the reconstitution in LUVs of an out-of-equilibrium metabolic network that regenerates ATP through the breakdown of L-arginine into L-ornithine27. The formation of ATP is coupled to the production of glycerol-3-phosphate (G3P), an important building block for phospholipid synthesis22,28. The metabolic pathway consists of two integral membrane proteins, an arginine/ornithine (ArcD) and a G3P/Pi antiporter (GlpT). In addition, three soluble enzymes (ArcA, ArcB, ArcC) are required for the recycling of ATP, and GlpK is used to convert glycerol into glycerol 3-phosphate, using the ATP from the breakdown of L-arginine, see Figure 1 for a schematic overview of the pathway. This protocol represents a good starting point for the future construction of even more complex reaction networks-for the synthesis of lipids or proteins or the division of cells. The lipid composition of the vesicles supports the activity of a wide variety of integral membrane proteins and has been optimized for the transport of diverse molecules into or out of the vesicles27,29,30.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66627/66627fig01v2.jpg" /> 
Figure 1: Overview of the pathway for ATP production and glycerol 3-phosphate synthesis and excretion. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In short, purified membrane proteins (solubilized in dodecyl-&#946;-D-maltoside, DDM) are added to preformed lipid vesicles that have been destabilized with Triton X-100, which allows the insertion of the proteins into the membrane. The detergent molecules are subsequently (slowly) removed by the addition of activated polystyrene beads, resulting in the formation of well-sealed proteoliposomes. Soluble components can then be added to the vesicles and encapsulated via freeze-thaw cycles, which traps the molecules in the process of membrane fusion. The obtained vesicles are highly heterogeneous and many are multilamellar. They are then extruded through a polycarbonate filter with a pore size of 400, 200, or 100 nm, which yields more uniformly sized vesicles; the smaller the pore size, the more homogeneous and unilamellar the vesicles but at the price of a smaller internal volume. Non-incorporated proteins and small molecules are removed from the external solution by size-exclusion chromatography. The proteoLUVs can be converted into micrometer size vesicles by gel-assisted swelling, and these proteoGUVs are then collected and trapped in a microfluidic chip for microscopic characterization and manipulation. Figure 2 shows a schematic overview of the full protocol.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66627/66627fig02v2.jpg" /> 
Figure 2: Overview of the protocol for reconstituting membrane proteins and encapsulating enzymes and water-soluble components in lipid vesicles of sub-micrometer (LUVs) and micrometer size (GUVs). <a href="https://www.jove.com/files/ftp_upload/66627/66627fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The reconstitution and encapsulation protocols work well and the functionality of the proteins is retained, but the proteoLUVs and proteoGUVs are heterogeneous in size. Microfluidic approaches31,32 allow the formation of micrometer-sized vesicles that are more homogeneous in size, but functional reconstitution of membrane proteins is generally not possible because residual solvent in the bilayer inactivates the proteins. The proteoLUVs range in size from 100 to 400 nm, and at low concentrations of enzymes, the encapsulation may lead to vesicles with incomplete metabolic pathways (stochastic effects; see Figure 3). LUVs are ideal for constructing specific metabolic modules, as shown here for the production of ATP and building blocks like G3P. Such proteoLUVs can potentially be encapsulated in GUVs and serve as organelle-like compartments for the host vesicles.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66627/66627fig03v2.jpg" /> 
Figure 3: Number of molecules per vesicle with a diameter of 100, 200, or 400 nm. (A) When the encapsulated proteins (enzymes, probes) are in the range of 1-10 &#181;M. (B) The reconstitution is done at 1 to 1,000, 1 to 10,000, and 1 to 100,000 membrane proteins per lipid (mol/mol). We make the assumption that molecules are encapsulated at the indicated concentrations and incorporated in the membrane at these protein-to-lipid ratios. For some enzymes, we have seen that they bind to membranes, which can increase their apparent concentration in the vesicles. Abbreviation: LPR = Lipid-Protein-Ratio <a href="https://www.jove.com/files/ftp_upload/66627/66627fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

저자 스포트라이트: 합성 세포 공학의 과제 해결

나노미터 및 마이크로미터 크기의 소포에서 평형을 벗어난 대사 네트워크 구축

In the Netherlands, the BaSyC consortium aims to construct synthetic cells from molecular components. The BaSyC consortium is composed of research groups ranging from molecular biology to chemistry, physics, and computational sciences. The goal of this consortium is to create a self-replicating system capable of autonomous growth to transmit information and to divide.And we currently focus on the building of molecular networks that are integrated towards more complex molecular systems. Recent developments in synthetic biology include the engineering of modules for cellular functions, integrating them into complex networks for applications in diagnostics, therapy, synthetic immunology, and adaptive materials. This progress spans groups in Europe, America, and Asia, advancing both top-down and bottom-up approaches to synthetic cell construction.Current technologies in our field include the engineering and evolutionary methods to construct and optimize metabolic networks. We use AI for molecular selection and advanced biochemical and biophysical techniques for component isolation and network formation. We also employ membrane reconstitution, physical encapsulation, and visualization via fluorescent spectroscopy and microscopy.With the integration of modules, the number of membrane proteins and enzymes increase enormously, which poses constraints on our reconstitution and encapsulation technologies. Stochastic encapsulation becomes an issue with sub-micron size vesicles, and the efficiency of forming micrometer-size vesicles decreases when multiple membrane proteins are reconstituted in the lipid membrane. In Groningen, we have developed metabolic networks for ATP productions and maintaining an out-of-equilibrium state through substrate feeding and product export.Additionally, we linked ATP recycling to the synthesis and excretion of lipid precursors, enabling secondary vesicles to produce lipids, which is a significant advancement in our field.

마이크로미터 크기의 소포를 위한 대사 네트워크 업스케일링

시작하려면 절단된 200마이크로리터 피펫 팁의 하단을 사전 제작된 미세유체 트래핑 장치에 삽입합니다. 0.2마이크로미터 필터가 장착된 주사기를 사용하여 피펫을 사용하여 400마이크로리터의 베타 카제인 용액을 칩의 입구 저장소에 주입합니다. 900G에서 6분 동안 칩을 원심분리합니다.이제 액체 레벨은 입구와 출구 모두에서 동일해야 합니다. 두 개의 대물렌즈 슬라이드에 스페이서의 윤곽을 그립니다. 고산소 플라즈마로 슬라이드를 1분 동안 청소하여 친수성으로 만듭니다.아가로스로 완전히 덮일 때까지 슬라이드 위에 낮은 겔화 온도의 아가로스를 추가합니다. 그런 다음 슬라이드를 90도 각도로 기울이고 여분의 아가로스를 조직에 배출합니다. 슬라이드를 섭씨 50도에서 30분 동안 놓습니다.1mm 프로브가 장착 된 휴대용 프로브 초음파 발생기를 사용하여 준비된 프로테오올리포좀을 초음파 처리합니다. 초음파 처리를 5 번 반복하기 전에 proteo SUV를 30 초 동안 얼음 위에 두십시오. 100 마이크로리터 주사기를 사용하여 프로테오 SUV의 0.5 마이크로리터 방울을 준비된 아가로스 겔에 증착합니다.약 10분 동안 질소 흐름을 사용하여 물방울을 건조시킵니다. 주사기와 바늘을 사용하여 측면의 작은 구멍을 통해 팽윤 용액을 준비된 챔버로 피펫팅하고 소포가 섭씨 22도에서 최소 30분 동안 부풀어 오르도록 합니다. 챔버를 단단한 표면에 두드려 겔에서 GUV를 채취합니다.현미경 검사를 위해 GUV를 포획하려면 현미경에 부동태화 미세유체 칩을 장착합니다. 결함을 확인한 후 구부러진 바늘로 튜브를 저장소 배출구에 연결하십시오. 다른 쪽 끝을 1밀리리터 주사기에 연결한 후 주사기를 분당 최대 유속이 10마이크로리터인 펌프에 장착합니다.저장소의 세척 버퍼를 새 매체로 교체하십시오. 최소 80 마이크로리터의 완충액 P로 세척하십시오.여분의 완충액을 제거한 후 프로테오 GUV를 저장소에 추가합니다. 충분한 GUV가 칩에 갇힐 때까지 시간이 지남에 따라 칩을 모니터링합니다.여분의 GUV 용액을 피펫팅합니다. 그런 다음 분당 0.1 내지 1 마이크로리터의 유속으로 세척 완충액 P를 첨가한다. 마지막으로, 충분한 양의 소포로 트랩의 위치를 파악합니다.현미경에 설정을 적용한 후 분당 0.5마이크로리터의 유속으로 저장소에 버퍼 R을 추가합니다. 낮은 겔링 온도의 아가로스 겔에서 건조된 프로테오 LUV를 재수화하면 마이크로미터 크기의 소포가 형성되었습니다. GUV를 채취하여 베타 카제인 부동태화 미세유체 장치에 가두었습니다.

upscaling-a-metabolic-network-for-micrometersized-vesicles

Research

JoVE Journal

Biochemistry

Upscaling a Metabolic Network for Micrometer&#45;Sized Vesicles

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

연구

생화학

Reconstitution of Purified Membrane Proteins into Preformed Liposomes

정제된 막 단백질을 미리 형성된 리포좀으로 재구성

Encapsulation of a Metabolic Network for ATP Recycling and Glycerol 3&#45;P Synthesis in Submicron&#45;Size Vesicles

서브미크론 크기 소포에서 ATP 재활용 및 글리세롤 3-P 합성을 위한 대사 네트워크 캡슐화