利用快速生长的细菌 Vibrio natriegens 进行无细胞蛋白表达

摘要

无细胞表达系统是用于重要蛋白质高通量合成与筛选的高效且经济的工具。本文介绍了利用Vibrio natriegens制备无细胞蛋白质表达系统的方法，可用于以质粒DNA、线性DNA和mRNA为模板快速生产蛋白质。

摘要

海洋细菌Vibrio natriegens因其快速生长的特性，作为新兴的微生物宿主在生物技术领域受到广泛关注。本文描述了一种使用常规实验室设备制备V. natriegens粗提细胞提取物的通用方案。该方案产率高，且特别优化以提高用户的可操作性并降低成本。无细胞蛋白合成（CFPS）可在96孔或384孔板中进行小规模（10 μL）批次反应，3小时内可重复地获得浓度超过> 260 μg/mL的超折叠绿色荧光蛋白（sfGFP）。总体而言，单个操作者可在1−2天内完成粗提细胞提取物的制备及无细胞蛋白合成。该方案可轻松整合到现有的蛋白合成流程中，从而推动生物制造和合成生物学应用的发展。

引言

无细胞蛋白质合成是一种多功能且经济高效的方法，用于表达有价值的蛋白质或肽^1,2,3,4。传统上，无细胞蛋白质合成采用Escherichia coli表达系统进行；然而，近年来越来越多的研究开始利用具有新颖特性的非模式生物作为无细胞表达的底盘系统^{5,6,7,8,9,10,11,12}。具有独特代谢特征的生物是替代E. coli无细胞系统的理想候选者。例如，海洋细菌Vibrio natriegens是目前已知生长最快的生物之一，其倍增时间少于10分钟¹³。这一特性使V. natriegens在科研与生物技术领域作为新兴的微生物宿主受到广泛关注^{14,15,16,17,18}。鉴于V. natriegens的快速生长速率与其高效的蛋白质合成能力和代谢效率密切相关^19,20,21，利用其细胞机制进行无细胞合成有望显著拓展快速蛋白质生产与高通量筛选的技术工具箱。

最近已证明一种无细胞的 V. natriegens 表达系统能够利用T7启动子在3小时内产生浓度超过> 260 μg/mL的超折叠绿色荧光蛋白（sfGFP）⁹。开发该方法的总体目标是为用户提供一种高度易得、成本效益高、可重复且产率高的无细胞蛋白表达系统，该系统可在短时间内使用常规实验室设备制备。本方案采用1 L摇瓶培养、脉冲式超声破碎细胞裂解，以及在96孔或384孔板中进行小规模批次反应，以实现最大程度的并行化和高通量筛选。通过添加3-磷酸甘油酸（3-PGA）作为能量来源，可实现长时间持续的蛋白质表达^8,22,23。成功完成本方案后，用户将能够利用V. natriegens粗细胞提取物以无细胞形式表达目标蛋白或一组蛋白。

从甘油保存菌种开始，收集光密度在 600 nm 处（OD₆₀₀）为 1.0 的 V. natriegens 菌体，制备粗细胞提取物。1 L 培养液可获得约 2−3 mL 提取物，足以进行超过 800 次含 25% 粗细胞提取物的无细胞反应。可使用质粒 DNA、线性 DNA 或 mRNA 模板表达蛋白质；然而，在使用野生型 V. natriegens 无细胞体系时，内源核酸酶对线性 DNA 模板的降解仍是主要缺点⁹。从 V. natriegens 培养物开始，单个操作者可在 1−2 天内完成可用于下游应用的蛋白质制备。

方案

1. Preparation of V. natriegens Crude Cell Extracts – Bacterial Culture

1. Prepare V. natriegens bacterial growth media LB-V2 as per Table 1. Sterilize the growth media by autoclaving. Allow media to reach to room temperature (RT). Store excess media at RT.
   CAUTION: Wear proper personal protection equipment (PPE) and consult lab specific instructions when operating an autoclave.
2. Use a glycerol stock of wild type V. natriegens to inoculate 3 mL of LB-V2 media. Grow overnight at 30 °C while shaking at 225 rpm.
3. Wash 1 mL of the overnight culture by centrifugation at 10,600 x g on a benchtop centrifuge for 1 min. Aspirate the supernatant without disturbing the resulting pellet and resuspend in 1 mL of fresh LB-V2 media.
4. In an autoclaved 4 L baffled Erlenmeyer flask with sterile cover, add 1 L of fresh LB-V2 growth media. Inoculate using 1 mL of washed overnight culture (1:1,000 dilution ratio). Grow culture at 30 °C while shaking at 225 rpm.
   NOTE: V. natriegens cultures can be scaled up or down while maintaining the 1:1,000 dilution ratio. For example, add 250 μL of washed overnight culture to 250 mL of fresh LB-V2 growth media in a 2 L baffled Erlenmeyer flask with sterile cover.
5. Monitor the culture’s OD₆₀₀ using a spectrophotometer. When culture reaches OD₆₀₀ = 1.0 ± 0.2, harvest culture via centrifugation at 3,500 x g for 20 min at 4 °C. Place pellet on ice.
   NOTE: V. natriegens grows rapidly, so close monitoring of the culture is necessary. Growth to OD₆₀₀ = 1.0 should take approximately 1.5−2 h at the 1:1,000 dilution ratio.
6. Aspirate the supernatant and immediately store the resultant bacterial pellet at -80 °C or directly proceed to cell lysis described in section 2.
   NOTE: This step is a good stopping point; however, it is recommended that the pellet be processed immediately or within 1−2 days for best results.

2. Preparation of V. natriegens Crude Cell Extracts – Cell Lysis

1. Prepare S30 lysis buffer as per Table 1 in sterile deionized (DI) water and adjust pH to 7.7 using glacial acetic acid.
   CAUTION: Glacial acetic acid should be handled with proper PPE when adjusting the pH.
2. Cool S30 lysis buffer to approximately 4 °C in a refrigerator or on ice before beginning the cell lysis procedure.
   NOTE: For a quick cool down, lysis buffer can be placed at -20 °C. Do not allow the buffer to freeze.
3. Place cell pellets on ice for 10−20 min or until completely thawed. Resuspend all pellets resulting from the same 1 L culture using 10 mL of cold S30 lysis buffer, then transfer suspension to a 50 mL tube. If pellets are not frozen in the freezer at -80 °C in step 1.6, proceed directly to resuspension.
   NOTE: Increase the volume of S30 lysis buffer used to initially resuspend pellets as needed.
4. Centrifuge suspension at 3,500 x g for 10 min at 4 °C. Aspirate the supernatant without disturbing the pellet. Wash the pellet a second time using 10 mL of cold S30 lysis buffer. Place pellet on ice.
5. In a cold room, add 500 μL of cold S30 lysis buffer to the pellet in the 50 mL tube. Using a wide-bore pipette tip, resuspend the pellet and carefully transfer the entire pellet suspension to a 2 mL tube.
   NOTE: If a wide bore pipette is not available, use a pair of scissors to cut the end of a 1 mL pipette tip to increase the bore for pellet transfer.
   1. Transfer as much pellet as possible without significantly increasing the volume; however, the pellet should be resuspended in enough liquid to be sonicated, as indicated by a homogenous suspension in the 2 mL tube. Do not overfill the 2 mL tube. The suspension should not exceed 1.5 mL; split into multiple tubes if necessary.
6. Keep the cell pellet on ice and work in a cold room. Fill a 600 mL beaker with ice and place a 2 mL tube holder on top of the ice.
   NOTE: It is helpful to place the tube holder near the side of the beaker, so the pellet suspension will be visible in the ice to monitor sonication progress (see Figure 1).
7. Vortex suspended pellet in 2 mL tube briefly to homogenize the cells, flick tubes to remove any cells on the bottom of the cap, and place into tube holder with cap open. Lower sonicator tip into the suspension so that it is just under the liquid surface.
8. Prepare the sonication set-up as depicted in Figure 2 using a sonicator and probe with a ⅛-inch tip diameter. Input the following settings into the sonicator control: 20 kHz frequency and 50% amplitude, pulse ON time: 10 s, pulse OFF time: 60 s.
9. Run the pulse sonication protocol for three cycles. If the total volume of the pellet suspension is > 500 μL, run the pulse sonication protocol six times.
   NOTE: Generally, 3−6 pulses are sufficient to lyse V. natriegens pellets; additional pulses may be required depending on sonicator. During sonication, use the adjustment knob platform to move the probe up and down to lyse any pellet that may have settled to the bottom of the tube. The tube can be removed from the holder and briefly vortexed to re-homogenize the suspension in between pulses. See discussion below for desired consistency of sonicated cells.
   CAUTION: Wear appropriate hearing protection when sonicator is active.
10. Following sonication, centrifuge crude cell extract at 16,000 x g for 30−45 min at 4 °C or until lysate is free of any cellular debris as depicted in Figure 3.
11. In a cold room, aliquot 50 μL of the resulting supernatants to new 2 mL tubes without disturbing the pellet.
    NOTE: Any debris that is transferred accidently into the new 2 mL tubes will drastically reduce extract’s capacity for high yielding protein synthesis.
    1. Optionally, set aside 10 μL of cell extract for post-lysis protein quantification in a separate 2 mL tube (see step 2.13 below).
12. Flash freeze crude cell extracts by placing tubes into a tube holder with a dipping string attached as depicted in Figure 4. Submerge tubes into a Dewar containing liquid nitrogen and immediately place in a freezer at -80 °C until use.
    CAUTION: Use appropriate PPE for handling liquid nitrogen including lab coat, safety glasses, face shield, cryogen apron, and gloves.
13. Optionally, quantify the total protein of the cell lysate using 10 μL set aside from step 2.11.1 by diluting sample 1:100 in 1x phosphate-buffered saline (PBS) and using any standard total protein quantification assay such as Bradford assay, bicinchoninic acid assay (BCA), etc.

3. Preparation of Cell-free Reaction Components

1. General reaction components
   1. Prepare working stocks of Mg-glutamate and K-glutamate in 50 mL tubes with sterile DI water at concentrations of 100 mM and 2000 mM, respectively.
   2. Prepare a working stock of 50% (w/v) polyethylene glycol (PEG)-8000 by adding 100 mL of sterile DI water to a 250 mL beaker. Place a small magnetic stirrer into the beaker. Weigh out 50 g of PEG-8000 and add to the 100 mL of water in the beaker.
   3. Place the beaker on a heated stir plate set to 100 °C and stir at 250 rpm until PEG-8000 is in solution. Allow PEG-8000 liquid mixture to cool before transferring to 50 mL tubes.
2. Energy solution master mix
   1. Prepare a 5 M solution of KOH by adding 140 g of KOH pellets to 500 mL of sterile DI water.
      CAUTION: Prepare this solution in a chemical hood and wear PPE when handling strong bases. The solution will become hot so the bottle cap must be loose to prevent pressure build-up. Allow the KOH solution to reach RT before using.
   2. Prepare a 1750 mM HEPES-KOH buffer by adding 20.85 g of HEPES to a 100 mL bottle. Slowly add sterile DI water until the volume reaches 40 mL. Vortex bottle to dissolve the HEPES. Use the 5 M KOH solution to adjust the pH to 8.0 and then bring the solution volume to 50 mL.
      CAUTION: KOH should be handled with proper PPE when adjusting the pH.
   3. Prepare the remaining 10x energy master mix stock components at the concentrations indicated in Table 2 in sterile DI water and place each stock on ice. Thaw the 100 mM ATP, GTP, CTP, and UTP stocks at RT and place on ice.
      NOTE: A thermomixer set to 37 °C and 350 rpm can be used to dissolve reagents into solution if necessary. Do not overheat or leave reagents on the thermomixer for an extended period of time.
   4. In a 15 mL tube, add each energy solution master mix component in accordance to the order and volume specified in Table 2. Vortex the solution after each component is added. This will make 5 mL of 10x energy solution master mix.
   5. Divide the 10x energy solution master mix into 200 μL aliquots in 2 mL tubes. Flash freeze each aliquot as performed in step 2.12. Immediately place them into the -80 °C freezer until use.
3. Amino acid master mix
   1. To prepare fresh 4x amino acid master mix, begin by thawing each amino acid stock at RT and then placing each on ice. Use a vortex and/or thermomixer set at 37 °C and 350 rpm to ensure all amino acid stocks are fully dissolved.
      NOTE: Cysteine may not fully dissolve; it can be added to the amino acid master mix as a suspension. Do not overheat or leave reagents on the thermomixer for an extended period of time.
   2. In a 15 mL tube, add the appropriate volume amino acids to sterile DI water so that final concentration of each is 8 mM in the following order: ALA, ARG, ASN, ASP, GLN, GLU, GLY, HIS, IIE, LYS, MET, PHE, PRO, SER, THR, VAL, TRP, TYR, LEU, and CYS. After adding each amino acid, vortex the master mix solution. The volumes listed in Table 2 will make up 2.4 mL of amino acid master mix.
   3. Divide the 4x amino acid master mix into 200 μL aliquots in 2 mL tubes. Flash freeze each aliquot as performed in step 2.12. Immediately place into a freezer at -80 °C until use.
4. Production of reaction-ready plasmid DNA template
   NOTE: Cell-free protein expression in this system has been optimized using the super folder green fluorescent protein (GFP) expression vector T7-pJL1-sfGFP (Table of Materials). It is recommended to use this plasmid as a control for cell-free reaction efficiency and the pJL1 backbone for the cloning and expression of other protein sequences. Other plasmid DNA templates can be used; however, it is important to note that transcription is controlled by a T7 promoter sequence and the presence of T7 RNA polymerase. A simple protocol for the large-scale production of any plasmid DNA template from transformed E. coli is described below.
   1. Purify desired vector using a plasmid purification kit as per manufacturer’s instruction (Table of Materials).
      NOTE: Concentrating the plasmid DNA template as much as possible is recommended to meet the tight volume constraints of the cell-free reaction. In general, aim for a 750−1500 ng/μL working stock.
5. Production of reaction-ready mRNA template
   NOTE: This section is optional. Protein expression has been tested using mRNA template generated from the in vitro transcription of the plasmid T7-pJL1-sfGFP by the listed T7 RNA polymerase (Table of Materials).
   1. Prepare mRNA template from plasmid DNA encoding the protein of interest using the in vitro transcription reaction components in Table 3. Incubate reactions for 1 h at 37 °C in a thermocycler.
      NOTE: It is recommended to perform 8−10x of these reactions in parallel to generate enough material.
   2. Pool all transcription reactions. Purify using an mRNA purification and concentrator kit as per manufacturer’s instruction (Table of Materials). Elute mRNA template into sterile DI water. Store mRNA template at -80 °C until use.

4. Performing Cell-free Protein Eexpression Reactions Using V. natriegens Crude Extract

1. Cell-free protein expression using plasmid or linear DNA template
   1. Remove 10x energy solution master mix and 4x amino acid master mix aliquots from the -80 °C freezer, thaw at RT, and place on ice. Remove the T7 RNA polymerase and RNase inhibitor stocks from the -20 °C freezer and place on ice. Thaw DNA template at RT and place on ice.
   2. Prepare a cell-free reaction master mix as per Table 4 by adding each component in the following order to a 2 mL tube on ice: amino acid master mix, energy solution master mix, Mg-glutamate, K-glutamate, DNA template, PEG-8000, T7 RNA polymerase, and RNase inhibitor. Gently flick the tube after each addition to the master mix.
      NOTE: If linear template is to be used for cell-free protein expression, add 5−10x more material as compared to plasmid template to obtain appreciable yields of protein.
   3. Remove V. natriegens crude cell lysate prepared in step 2.12 from the -80 °C freezers and place on ice for 10−20 min until thawed. Add the appropriate volume crude cell extract to the cell-free reaction master mix as per Table 4 and gently mix by flicking or pipetting up and down.
   4. End-point cell-free protein expression using thermocycler
      1. Pipette 10 μL of the cell-free reaction master mix to the bottom of a 96- or 384-well PCR plate. In between each transfer to the PCR plate, mix the master mix by flicking the tube gently.
         NOTE: Cell-free reaction master mix should be well mixed at all times to maximize reaction reproducibility and cell-free protein expression in all samples.
   5. Briefly centrifuge the plate at 1,000 x g for 10 s to pool any master mix that may have become stuck on the sides of the wells. Seal the wells with a plate adhesive to prevent evaporation and then place the PCR plate into a thermocycler set at 26 °C with a heated lid set at 105 °C.
      NOTE: The even heat distribution and heated lid of a thermocycler greatly improves protein expression yields.
   6. Incubate the cell-free reactions for a minimum of 3 h. After incubation, expressed proteins can be purified, quantified, and used for downstream processes.
      NOTE: Expressed proteins can be directly quantified in the cell-free reaction using a method of the user’s choice. For example, fluorescent proteins can be quantified using an external standard curve or radioactivity can be measured if using a radiolabeled amino acid in the cell-free reaction. UV-visible spectroscopy or total protein assays are generally not recommended for directly measuring protein production in cell-free reactions without an initial purification.
   7. Alternatively, monitor cell-free protein expression kinetics using a plate reader.
      1. Pipette 10 μL of the cell-free reaction master mix to the bottom of a black 384-well assay plate with clear glass bottoms. Keep the assay plate on ice or work in a cold room while adding the master mix to ensure that the full kinetic profile is obtained. Seal the wells with a clear plate adhesive to prevent evaporation and then place the assay plate into a plate reader set at 26 °C.
   8. Incubate the cell-free reactions for 3−6 h while monitoring the appropriate fluorescent excitation/emission wavelengths corresponding to the expressed protein. For example, monitor at Ex/Em = 485 nm/528 nm for sfGFP.
2. Cell-free protein expression using mRNA template
   1. Thaw mRNA template prepared in step 3.5.2 at RT and place on ice.
   2. Perform step 4.1.1 through step 4.1.6 as previously specified for linear or plasmid DNA template using Table 4 to prepare an alternative cell-free reaction master mix for mRNA template.

5. Calibration of V. natriegens Cell-free Reactions with sfGFP

NOTE: This section is optional. The optimal cell-free reaction ion concentration can vary slightly for each crude extract preparation based on the conditions used for cell lysis. Consider performing the optional cell-free reaction ion calibration protocol described below using sfGFP if reaction yields are significantly lower than expected (concentrations < 1.0 μg/mL).

1. Prepare the Mg²⁺ and K⁺ ion calibration solutions as specified in Table 5 in sterile DI water from the 100 mM Mg-glutamate and 2,000 mM K-glutamate stocks prepared in step 3.1.1. Place calibration solutions on ice until needed.
2. Following the calibration map in Table 6, pipette 1 μL of the Mg-glutamate and 2 μL of K-glutamate ion calibration solutions into the appropriate wells in a 384-well PCR plate.
   1. The order of operations of this step is as follows: Pipette the Mg-glutamate ion calibration solution into the bottom of each well followed by the K-glutamate ion calibration solution onto the side of well without touching the liquid already present in the wells.
   2. Seal the wells by placing an adhesive on top of the plate to prevent evaporation while preparing the calibration cell-free reaction master mix. Gently tap the 384-well plate to mix the Mg- and K-glutamate calibration solutions.
      NOTE: A repeater pipette is highly recommended for completing this step reproducibly and quickly.
3. Prepare the calibration cell-free reaction master mix as specified in Table 5 using T7-pJL1-sfGFP plasmid DNA template in a 2 mL tube. Add each component in the following order to the master mix: amino acid master mix, energy solution master mix, T7-pJL1-sfGFP DNA template, PEG-8000, T7 RNA polymerase, and RNase inhibitor. Gently flick the tube to mix after each component addition.
   NOTE: Do not add Mg²⁺ or K⁺ to the calibration cell-free master mix.
4. Remove the adhesive from the plate. Carefully pipette 7 μL of the calibration cell-free reaction master mix on to the sides of the wells without touching the mixed ion calibration solution already present in the wells. Reseal the wells with the adhesive and gently tap the 384-well PCR plate to mix the cell-free master mix with the ion calibration solution.
   NOTE: A repeater pipette is highly recommended for completing this step reproducibly and quickly.
5. Briefly centrifuge the plate 1,000 x g for 10 s to pool any unmixed liquid that may have become stuck on the sides of the wells. Place the 384-well plate into a thermocycler set at 26 °C with a heated lid set at 105 °C.
6. Incubate the cell-free reactions for 3 h. After incubation, transfer the contents of each well to a black 384-well assay plate with glass bottoms with a multi-channel pipette and read the fluorescence of sfGFP at Ex/Em = 485 nm/528 nm using a plate reader to determine the ion concentration combination that yields the highest amount of protein.

结果

使用V. natriegens无细胞表达系统进行蛋白质生产的所述方案，从培养物接种到获得可用于下游应用的蛋白质，单个用户可在1−2天内完成。粗细胞提取物和主混合物储备液的制备占用了大部分时间；然而，一旦制备完成，大多数大量试剂可长期储存（表7），并按需使用，从而缩短完成该方案所需的时间。

所述表达系统最适合使用质粒DNA模板或通过体外转录反应生成的mRNA。尽管线性DNA也可用作蛋白质生产的模板，但其产生的蛋白量显著较低。例如，在26 °C的最佳反应条件下，单个10 μL的无细胞反应体系在3小时内使用0.3 pmol质粒DNA模板可产生> 260 μg/mL的sfGFP，或使用14 pmol mRNA转录本可产生相当的> 125 μg/mL的sfGFP（图5A,B）。然而，使用0.3 pmol线性DNA产生的蛋白量显著更低（< 20 μg/mL）。对于每种模板类型，大部分蛋白在1−1.5小时内生成；但建议反应时间至少运行3小时。sfGFP的无细胞反应浓度通过纯化的sfGFP标准曲线进行线性回归测定，荧光检测激发/发射波长为Ex/Em = 485 nm/528 nm。

蛋白质的产量显著受到钾离子和镁离子（分别为 K⁺ 和 Mg²⁺）浓度的影响。在优化条件下，发现 Mg²⁺ 和 K⁺ 离子的最适浓度分别为 3.5 mM 和 80 mM（图 6A,B）。若离子浓度偏离最适水平，可能导致无细胞表达系统产生蛋白质的能力下降，产量显著降低。如果反应产量明显低于预期，可能需要进行额外的校准。第 5 节中描述了一种可选的离子校准方案，该方案可在一定程度上补偿因不同细胞裂解设备和条件导致的粗提细胞提取物的差异性。

图1：超声平台上的烧杯和试管架的特写图。 请点击此处查看该图的放大版本。

图2：制备粗细胞提取物的超声破碎设备装置。 请点击此处查看该图的放大版本。

图 3：超声处理和离心后沉淀物的观察。 请点击此处查看此图的放大版本。

图 4：用于提取物储存的快速冷冻浸渍装置。 请点击此处查看此图的放大版本。

图5：使用不同类型模板进行V. natriegens无细胞蛋白质合成的代表性结果。（A）在最佳反应条件下，以等摩尔浓度的质粒和线性DNA模板（0.3 pmol）以及不同浓度的mRNA模板，在10 μL反应体系中于26 °C孵育180分钟后，通过Ex/Em = 485 nm/528 nm处测定纯化的sfGFP标准曲线，确定无细胞体系中sfGFP的浓度。（B）在相同最佳反应条件和10 μL反应体积下，每隔3分钟于Ex/Em = 485 nm/528 nm处检测sfGFP生成的动力学实验，所用模板为等摩尔浓度的质粒和线性DNA以及不同浓度的mRNA。终点和动力学实验均使用除模板外其余组分齐全的无细胞反应作为空白对照进行校正。数据以均值±标准差表示（n = 3）。请点击此处查看该图的放大版本。

图 6：离子浓度校准的代表性结果。 （A）在 V. natriegens 无细胞反应体系中添加递增浓度的 Mg²⁺，于 26 °C 孵育 180 分钟，反应体积为 10 μL。所有 Mg²⁺ 浓度条件下，K⁺ 总浓度保持在 160 mM。通过纯化 sfGFP 的标准曲线测定无细胞体系中 sfGFP 的浓度，检测波长为激发/发射波长（Ex/Em）= 485 nm/528 nm。（B）在 V. natriegens 无细胞反应体系中添加递增浓度的 K⁺，于 26 °C 孵育 180 分钟，反应体积为 10 μL。所有 K⁺ 浓度条件下，Mg²⁺ 总浓度保持在 3.5 mM。两次校准实验中，均使用缺少模板但包含其余所有组分的无细胞反应作为空白对照。数据以均值 ± 标准差表示（n = 3）。本图经 Wiegand 等人⁹ 修改后重印。转载自 Wiegand, D.J., Lee, H.H., Ostrov, N., Church, G.M. 建立一种无细胞的 Vibrio natriegens 表达系统。ACS Synthetic Biology. 7 (10), 2475−2479 (2018)。版权所有 © 2018 美国化学学会。请点击此处查看本图的放大版本。

表 1 A	LB-V2 细菌培养基的制备
组分	用量 (g)	终浓度 (mM)	终体积 (L)
LB 肉汤（Miller）	25		1
NaCl	11.69	200
MgCl2	2.20	23.1
KCl	0.31	4.2
表 1 B	S30A 裂解缓冲液的制备
组分	用量 (g)	终浓度 (mM)	终体积 (L)
Tris 溶液 (pH 8.0) - 1 M (mL)*	25	50	0.5
Mg-谷氨酸盐	2.72	14
K-谷氨酸盐	6.10	60
二硫苏糖醇 (DTT) - 1 M (mL)*	1	2

表1：制备1 L LB-V2细菌培养基和0.5 L S30A细胞裂解缓冲液所需的试剂。请点击此处下载Excel文件。 

	10倍能量溶液主混合液的配制
组分	储存浓度 (mM)	终浓度 (mM)	体积 (µL)	终体积 (µL)
HEPES-KOH pH 8	1750	500	1428.57	5000
ATP	100	15	750.00
GTP	100	15	750.00
CTP	100	9	450.00
UTP	100	9	450.00
来自E. coli MRE 600 的 tRNA (mg/mL)*	100	2	100.00
辅酶A水合物	200	2.6	65.00
NAD	200	3.3	82.50
cAMP	650	7.5	57.69
亚叶酸	100	0.7	35.00
亚精胺	1600	10	31.25
3-PGA	2000	300	750.00
无菌去离子水			49.99
	4倍氨基酸主混合液的配制
组分	储存浓度 (mM)	终浓度 (mM)	体积 (µL)	终体积 (µL)
ALA	168	8	114.3	2400
ARG	168	8	114.3
ASN	168	8	114.3
ASP	168	8	114.3
GLN	168	8	114.3
GLU	168	8	114.3
GLY	168	8	114.3
HIS	168	8	114.3
IIE	168	8	114.3
LYS	168	8	114.3
MET	168	8	114.3
PHE	168	8	114.3
PRO	168	8	114.3
SER	168	8	114.3
THR	168	8	114.3
VAL	168	8	114.3
TRP	168	8	114.3
TYR	168	8	114.3
LEU	140	8	137.1
CYS	168	8	114.3
无菌去离子水			91.4

表2：配制5 mL 10倍能量溶液主混合液和2.4 mL 4倍氨基酸主混合液所需的试剂。  请点击此处下载Excel文件。

	T7 RNA 聚合酶体外转录反应
组分	储存液浓度 (mM)	终浓度 (mM)	单次反应用量 (µL)	50 次反应用量 (µL)
10x RNAPol 反应缓冲液			1.00	50
ATP	100	0.5	0.10	5
GTP	100	0.5	0.10	5
CTP	100	0.5	0.10	5
UTP	100	0.5	0.10	5
DNA 模板 (ng/µL)*	1000	500	0.50	25
T7 RNA 聚合酶	200	2.6	2.00	100
小鼠来源 RNase 抑制剂	200	3.3	0.50	25
无菌去离子水			15.60	780
反应体积 (µL):	20

表3：体外转录生成mRNA的组分。  请点击此处下载Excel文件。

	无细胞反应主混合液
组分	储存浓度 (mM)	终浓度 (mM)	单次反应用量 (µL)	50次反应用量 (µL)
提取物 (%) *		25	2.50	125.00
Mg-谷氨酸盐	100	3.5	0.35	17.50
K-谷氨酸盐	2000	80	0.40	20.00
4x 氨基酸主混合液	8.0	2	2.50	125.00
10x 能量溶液主混合液			1.00	50.00
质粒DNA (ng/µL) *	1000	500	0.50	25.00
50% PEG-8000 (%) *	50	2	0.40	20.00
T7 RNA聚合酶			1.00	50.00
鼠源RNase抑制剂			0.10	5.00
无菌去离子水			1.25	62.50
反应体积 (µL):	10
	用于mRNA模板的替代无细胞反应主混合液
组分	储存浓度 (mM)	终浓度 (mM)	单次反应用量 (µL)	50次反应用量 (µL)
提取物 (%) *		25	2.50	125.00
Mg-谷氨酸盐	100	3.5	0.35	17.50
K-谷氨酸盐	2000	80	0.40	20.00
4x 氨基酸主混合液	8.0	2	2.50	125.00
10x 能量溶液主混合液			1.00	50.00
mRNA模板 (ng/µL) *	2000	4000	2.00	100.00
50% PEG-8000 (%) *	50	2	0.40	20.00
鼠源RNase抑制剂			0.10	5.00
无菌去离子水			0.75	37.50
反应体积 (µL):	10

表4：用于DNA模板和mRNA模板的优化版V. natriegens无细胞反应主混合液的组分。  请点击此处以下载Excel格式文件。

Mg2+ 标准曲线配制		单次反应用量 (µL)	单次反应用量 (µL)	100 次反应用量 (µL)	100 次反应用量 (µL)
终浓度 (mM)	储备液	100 mM Mg-Glu	diH20	100 mM Mg-Glu	去离子 H2O
2.5	0	0.00	1.00	0	100
3.5	1	0.10	0.90	10	90
4.5	2	0.20	0.80	20	80
5.5	3	0.30	0.70	30	70
反应体积 (µL):	10
K+ 标准曲线配制		单次反应用量 (µL)	单次反应用量 (µL)	100 次反应用量 (µL)	100 次反应用量 (µL)
终浓度 (mM)	储备液	2000 mM K-Glu	diH20	2000 mM K-Glu	去离子 H2O
40	30	0.15	1.85	12	148
80	70	0.35	1.65	28	132
160	150	0.75	1.25	60	100
320	310	1.55	0.45	124	36
反应体积 (µL):	10
	离子标准曲线无细胞反应主混合液
组分	储备液浓度 (mM)	终浓度 (mM)	单次反应用量 (µL)	50 次反应用量 (µL)
提取物 (%)*		25	2.50	125.00
Mg-谷氨酸盐	可变	可变	1.00	50.00
K-谷氨酸盐	可变	可变	2.00	100.00
4x 氨基酸主混合液	8.0	2	2.50	125.00
10x 能量溶液主混合液			1.00	50.00
质粒 DNA (ng/µL)*	1000	250	0.25	12.50
50% PEG-8000 (%)*	50	2	0.40	20.00
T7 RNA 聚合酶			1.00	50.00
小鼠 RNase 抑制剂			0.10	5.00
无菌去离子水			0.00	0.00
反应体积 (µL):	10

表5：离子浓度校准母液混合物。  请点击此处下载Excel文件。

CF 离子校准图		40 mM K+			80 mM K+			160 mM K+			320 mM K+
		1	2	3	4	5	6	7	8	9	10	11	12
2.5 mM Mg2+	A
3.5 mM Mg2+	B
4.5 mM Mg2+	C
5.5 mM Mg2+	D

表6：离子校准图。  请点击此处下载Excel文件。

	CF 组分的储存条件与保质期
组分	储存位置	保质期
无菌 LB-V2 培养基	4 °C	3-6 个月
V. natriegens 粗制细胞提取物	-80 °C	1-3 周
100 mM Mg-Glutamate	室温	6 个月
2000 mM K-Glutamate	室温	6 个月
50% PEG-8000	室温	6 个月
10x 能量溶液母液混合物	-80 °C	3-6 个月
4x 氨基酸母液混合物	-80 °C	3-6 个月
质粒/线性 DNA 模板	-20 °C	6-12 个月
mRNA 模板	-80 °C	3-4 周

表7：无细胞物质的储存条件和保质期。  请点击此处下载Excel文件。

讨论

本方案已针对野生型 V. natriegens 及含有V2盐的LB培养基（材料表）进行了优化。其他 V. natriegens 菌株也可采用类似方法培养，以制备用于无细胞反应的粗细胞提取物；但使用这些菌株时需要对该方案进行进一步优化。此外，本无细胞蛋白表达系统已采用3-磷酸甘油酸（3-PGA）作为主要的能量再生来源进行优化。也可使用其他能量再生来源；但可能需要对试剂进行优化和校准，才能实现高效的蛋白表达^10,11。

特别注意本方案中的几个关键步骤，可确保获得最大提取效率，从而实现高产率的无细胞蛋白质合成。首先，必须使用处于对数生长中期收获的 V. natriegens 细胞培养物制备粗提细胞提取物；当培养物的 OD₆₀₀ 达到 1.0 ± 0.2 时，蛋白质产量最高。尽管在不同光密度下收获的细胞均可用于无细胞蛋白质合成，但我们先前的研究发现，处于对数生长期的细胞所获得的蛋白质产量显著更高⁹。光密度对粗提细胞提取物性能的影响与在分批培养条件下生长的其他细胞来源的无细胞表达系统中报道的结果一致¹。由于 V. natriegens 生长速度较快，必须密切监测培养物的光密度。通常情况下，采用本方案时，V. natriegens 培养物可在 1−1.5 小时内稳定达到 OD₆₀₀ 为 1.0；然而，个体培养条件（如使用带挡板与不带挡板的锥形瓶，这会影响通气效果，或空气浴与水浴培养，这会影响培养温度的升高速率和稳定性）可能会改变其生长时间。此外，通常建议在 1 L 带挡板锥形瓶中培养至少 250 mL 的 V. natriegens，以确保收获时获得足够大的细胞沉淀，便于后续操作和转移。这将显著提高粗提细胞提取物制备的成功率，并增加从单次沉淀中获得的提取物体积。在进行小规模制备时，可相应调整培养条件和试剂用量。对于大规模发酵，则可能需要进一步优化培养条件。最后，为确保高蛋白质产量，细胞沉淀必须在收获后立即处理，或在 -80 °C 下保存 1−2 天内完成处理。

通过脉冲超声处理对细胞沉淀进行充分裂解，对无细胞蛋白表达的成功至关重要，对于初次使用者而言，这通常是本实验方案中最困难的部分。通常情况下，裂解充分的沉淀会产生大量无碎片的澄清液体提取物。提取物应略带黏性，但在分装至储存管并投入液氮速冻前，仍可轻松地用移液器转移。图3展示了裂解良好（图3A）与裂解不充分（图3B）的沉淀在裂解后离心步骤中的对比示意图。细胞裂解完全的一个重要指标是，通过总蛋白测定法（步骤2.13）测得的粗细胞提取物总蛋白浓度> 20 mg/mL。过度超声或粗提取物温度过高会损伤细胞内的分子机器，而此类损伤若不进行无细胞反应则无法察觉。因此，在将大量时间和精力投入下游蛋白表达应用之前，使用对照反应检测提取物的活性极为重要。尽管不同超声设备可能需要进一步优化条件，但本文所述的脉冲超声步骤在我们的实验中具有高度可重复性。

利用PCR扩增、限制性内切酶消化或商业基因合成获得的线性DNA模板，可显著提高无细胞表达系统中蛋白质的高通量和快速生产能力²⁴。尽管已有利用PCR扩增的线性模板进行蛋白质生产的报道，但在等摩尔浓度条件下，其产量比使用质粒DNA模板的反应低约13.5倍⁹。这主要是由于线性DNA模板不稳定，可能被V. natriegens粗细胞提取物中存在的内源性核酸酶降解所致。虽然此前已使用λ噬菌体蛋白GamS来保护线性DNA模板^24,25，但研究发现其与V. natriegens提取物不兼容⁹。此外，尽管提高线性DNA模板的浓度可能有助于获得更高的蛋白产量，但其在粗细胞提取物中快速降解的问题仍是一个主要障碍。

克服线性DNA模板降解的一种方法可能是在线性DNA体外转录生成的mRNA模板基础上补充无细胞反应体系。另一方面，使用RNase抑制剂可显著防止mRNA转录本的降解，并可在10 μL无细胞反应体系中获得可观的蛋白质产量（图5A，B）。通过TA连接、TOPO克隆、Golden Gate组装或其他重组方法将线性DNA克隆至环状模板中，也可避免模板降解。然而，要利用线性DNA模板实现高效的蛋白质表达，仍需进一步开发抑制核酸酶活性的方法。

迄今为止，已有多种不同的方法被提出用于制备无细胞蛋白表达所需的粗提物^9,10,11,26。在开发本实验方案时，我们力求最大限度地提高用户的可及性，降低总体成本，并减少耗时的操作步骤。例如，本方法仅通过简单的两步超声-离心处理即可获得较高的蛋白产量，无需使用细胞匀浆器、长时间透析步骤或跑脱反应。该方法可在短时间内轻松完成，且不需要高水平的实验室操作技能。因此，该方案有助于推动无细胞表达成为转化型学术研究和工业流程设计中的常规技术。

本方案拓展了可用于研究和利用V. natriegens（一种具有独特生物学特性的非模式生物）的工具集。通过采用半连续或全连续的无细胞反应体系，可实现能量再生、氨基酸的持续补充以及代谢废物的清除，从而获得更高的蛋白质产量^3,5,27。此外，对野生型V. natriegens进行基因改造，构建DNA酶或RNA酶缺陷型菌株，去除有害且竞争性的代谢通路，并表达额外的tRNA，可显著提升该系统中蛋白质的表达水平^28,29。随着对其快速生长机制相关生物学基础的深入解析，V. natriegens无细胞系统的进一步开发有望加速生物制造能力，并实现治疗性多肽、小分子及合成材料的高效表达。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
15 mL 离心管	Corning	352196
2 mL 离心管	Eppendorf	22363352
384 孔黑色检测板	Corning	3544
384 孔 PCR 板	Eppendorf	951020702
50 mL 离心管	Corning	352070
96 孔 PCR 板	Eppendorf	30129300
3',5'-环腺苷酸单钠盐一水合物	Sigma	A6885
5'-三磷酸腺苷 - 100 mM	NEB	N0450L
Applied Biosystems Veriti 384 孔热循环仪	ThermoFisher	4388444
检测板封板膜	BioRad	MSB1001
β-烟酰胺腺嘌呤二核苷酸水合物（NAD）	Sigma/Roche	10127965001
辅酶 A 水合物	Sigma	C4283
5'-三磷酸胞苷 - 100 mM	NEB	N0450L
D-(-)-3-磷酸甘油酸二钠盐（3-PGA）	Sigma	P8877
杜瓦瓶 - 4 L	ThermoScientific	10-194-100C
二硫苏糖醇溶液 - 1 M	Sigma	42816
亚叶酸钙盐水合物	Sigma	47612
冰乙酸	Sigma	A6283
5'-三磷酸鸟苷 - 100 mM	NEB	N0450L
HEPES	Sigma	H3375
L-谷氨酸半镁盐四水合物	Sigma	49605
L-谷氨酸钾盐一水合物	Sigma	G1149
LB 培养基（Miller）	Sigma	L3522
氯化镁（MgCl2）	Sigma	M8266
质粒 pJL1-sfGFP	Addgene	69496
Plasmid Plus Maxi 试剂盒	Qiagen	12963
聚乙二醇（PEG）-8000	Sigma	89510
氯化钾（KCl）	Sigma	P9333
氢氧化钾颗粒	Sigma/Roche	1050121000
Q125 超声波破碎仪及带 1/2 英寸探头的 CL-18 探针	Qsonica	4422
RNA 纯化与浓缩试剂盒	Zymo	R1013
鼠源 RNase 抑制剂	NEB	M0314
RTS 氨基酸筛选试剂盒	Biotechrabbit	BR1401801
氯化钠（NaCl）	Sigma	S7653
亚精胺	Sigma	S0266
T7 RNA 聚合酶	NEB	M0251
Tris 溶液（pH 8.0）- 1 M	Invitrogen	AM9856
来自 E. coli MRE 600 的 tRNA	Sigma/Roche	10109541001
5'-三磷酸尿苷 - 100 mM	NEB	N0450L
Vibrio natriegens （野生型）冻干菌种	ATCC	14048