Jun 2020



Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins

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Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins) with multiple intramolecular disulphide bonds which are typically difficult to express in a soluble and folded state in the reducing environments found inside a cell. In some cases, the inclusion of a recombinant disulphide isomerase DsbC further enhances the expression levels of correctly folded hydrophobins. Using this protocol, we can achieve milligram levels of protein expression per ml of reaction. While our target proteins are the fungal hydrophobins, it is likely that this protocol with some minor variations can be used to express other proteins with multiple intramolecular disulphide bonds in a natively folded state.

Graphic abstract:

Figure 1. Workflow for cell-free protein expression and single-step purification using affinity chromatography. A. E. coli S30 lysate prepared as described in Apponyi et al. (2008) can be stored for up to several years at -80°C without any loss of activity in our experience. B. The S30 lysate, plasmid DNA that encodes for the protein of interest along with an affinity tag and components required for transcription and translation are added to the reaction mix. Following a single-step protein purification, the protein of interest can be isolated for further use.

Keywords: Hydrophobins (疏水蛋白), Disulphide bonds (二硫键), Protein synthesis (蛋白质合成), Cell-free (无细胞的), Protein production (蛋白质生产), DsbC (DsbC)


The bacterial cytosol microenvironment is home to thousands of diverse reactions and is tightly regulated in a reducing redox state. This poses challenges in recombinant bacterial protein overexpression as the reducing environment can prevent the correct formation of critical disulphide bonds in proteins that require them for structure and/or function (Kadokura and Beckwith, 2010). As such, these proteins are often expressed by E. coli into insoluble inclusion bodies (Berkmen, 2012) that renders them unusable. In favorable cases, proteins from inclusion bodies can be refolded in vitro after solubilizing in strong denaturants (Qin et al., 2015). However, this is typically a lengthy and reagent consuming process and the yield of the correctly refolded protein is variable.

One alternative to conventional recombinant bacterial overexpression is cell-free synthesis, where proteins can be expressed using the bacterial transcription and protein production machinery, but without the confines of a cell (Apponyi et al., 2008; Ozawa and Loh, 2014; Gao et al., 2019; Dondapati, 2020). Cell-free protocols are particularly useful in the expression of cysteine-rich proteins, as the redox environment can be controlled by simply changing the ratios of reduced and oxidized glutathione (GSH:GSSG) and varying the concentration of reducing agents such as dithiothreitol (DTT). After optimization, the yield of protein with correctly formed disulphide bonds suitable for downstream experiments (e.g., binding studies, structural and biophysical characterisation) can be greatly increased.

Beyond proteins with disulphide bonds, cell-free protein synthesis systems can hold additional advantages over traditional recombinant protein production such as the ability to readily incorporate unnatural amino acids (Gao et al., 2019; Dondapati et al., 2020). In addition, proteins that are too toxic for a host cell can be expressed in a cell-free environment as there is no requirement to maintain cell viability and this has allowed high throughput production and screening for membrane proteins, enzymes, and other therapeutics (Khambhati et al., 2019).

Our optimized cell-free expression protocol allows direct control of the redox conditions in the reaction mixture and demonstrates a distinct advantage over recombinant expression for a class of proteins known as the fungal hydrophobins (Siddiquee et al., 2020). This class of proteins is characterized by four disulphide bonds and the correct bonding pattern is required for hydrophobins to self-assemble into amphipathic layers at hydrophilic:hydrophobic interfaces (Sunde et al., 2008; Ball et al., 2020). These unique assemblies have suggested a range of biotechnological applications from drug delivery systems to coating implants and the engineering of surfaces to increase biocompatibility (Wosten and Scholtmeijer, 2015; Berger and Sallada, 2019). While many hydrophobins can be overexpressed in E. coli to a very high yield, the protein is almost exclusively found misfolded within inclusion bodies and cannot be used for downstream applications. Therefore, a long process of in vitro refolding and multi-step purification is typically required to separate the correctly folded protein from the partially folded and misfolded species (Kwan et al., 2006; Pille et al., 2015; Pham et al., 2016). To overcome this problem, we have developed a cell-free expression system for hydrophobin production based on the published S30 E. coli lysate (Apponyi et al., 2008).

The S30 lysate is one of the most used extracts in cell free synthesis of proteins and contains the cellular transcriptional and translational machinery. A Pubmed search with “S30 cell free” as search terms gives >150 articles. While the S30 lysate can be purchased commercially (e.g., from Promega), it can be produced readily in laboratories that perform recombinant protein expression and purification at a fraction of the cost of commercially available cell-free expression kits. This cell-free reaction involves incubating the lysate and other key cofactors with a gene of interest cloned downstream of a constitutive promoter in a dialysis set up. The dialysis set up allows continuous exchange of lower molecular reagents (e.g., amino acids, rNTPs, ATP) and offers higher protein yields per unit of components that are the more expensive or time-consuming to prepare (e.g., transcription and translation machinery in the S30 extract as well as plasmid DNA).

To aid purification, we have cloned the gene into the vector pET-MCSIII that has a hexa-histidine affinity tag but lacks the lac operator (Neylon et al., 2000). The protocol presented herein can be easily amended to enable the expression of other proteins with disulphide bonds. The apparatus used in this protocol can be easily prepared using disposable plastic tubes commonly used in most scientific laboratories and can be scaled up as required. The efficiency and low cost of this set up allow multiple proteins (e.g., a protein and its mutants) to be produced in parallel cell-free reactions using one preparation of dialysis buffer. Isotopically labeled proteins can also be easily produced by substituting in labeled amino acids in the reaction mixture. In some cases, this has allowed Nuclear Magnetic Resonance (NMR) and mass spectroscopy experiments to be carried out on the protein of interest within one day of setting up the reaction and without the need for further purification.

Materials and Reagents

  1. Eppendorf tubes 1.5 ml (Thermo Fisher Scientific, catalog number: 69715)

  2. Eppendorf tubes 0.5 ml (Eppendorf, catalog number: 0030121023)

  3. Greiner Bio-One 50 ml tubes

  4. Pipette tips

  5. S30 Lysate prepared according to Apponyi et al. (2008)

  6. rNTP (Sigma-Aldrich, catalog numbers: A6559, C8552, G3776, U1006)

  7. HEPES (Sigma-Aldrich, catalog number: 15630106)

  8. Folinic acid (Sigma-Aldrich, catalog number: 47612)

  9. cAMP (Sigma-Aldrich, catalog number: A6885)

  10. NH4OAc (Sigma-Aldrich, catalog number: A1542)

  11. ATP (Sigma-Aldrich, catalog number: A1852)

  12. Creatine phosphate (Sigma-Aldrich, catalog number: 27920)

  13. Potassium glutamate (Sigma-Aldrich, catalog number: G1501)

  14. Creatine kinase (Sigma-Aldrich, catalog number: C9983)

  15. Mg(OAc)2 (Sigma-Aldrich, catalog number: M5661)

  16. tRNA (Sigma-Aldrich, catalog number: R8759)

  17. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)

  18. L-Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251)

  19. L-Glutathione oxidised (GSSG) (Sigma-Aldrich, catalog number: G4376)

  20. 20 amino acids (Sigma-Aldrich)

  21. 10,000 MWCO SnakeSkin® dialysis membrane (Thermo Fisher Scientific, catalog number: 88243). Please note to choose a membrane cutoff that is at least three times smaller than the size of the expected protein product

  22. NuPAGETM 4-12% Bis-Tris Protein Gel (Invitrogen, catalog number: NP0323PK2) or any SDS-PAGE gel that is suitable for the detection of the protein of interest

  23. MES buffer (Invitrogen, catalog number: NP0002) or other compatible buffers for the running the SDS-PAGE gel

  24. NuPAGETM LDS Sample Buffer (4×) (Thermo Fisher Scientific, catalog number: NP0007)

  25. PBS (Sigma-Aldrich, catalog number: P4417)

  26. Urea (Sigma-Aldrich, catalog number: U5378)

  27. Recombinant disulphide isomerase DsbC (Note 2)

  28. Plasmid or PCR-amplified fragment encoding for the protein of interest (Note 3)


Standard laboratory equipment commonly available in molecular biology and protein laboratories, such as pipettes, fridges, magnetic stirrers have not been included. Equipment that have similar specifications to those listed below can also be used.

  1. Scalpel

  2. NanoDropTM UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: ND-2000)

  3. Bunsen burner

  4. Incubator (Eppendorf, model: Innova 44; M1282-0002)

  5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus (ThermoFisher Scientific, catalog number: A25977)


  1. Microsoft Excel


Cell-free (in vitro) protein synthesis

The cell-free reaction involves incubating the S30 lysate with the DNA construct, encoding the gene of interest under a constitutive promoter, and the necessary cofactors in a dialysis setting. For more details about the DNA construct, please see Note 3.

    When testing new constructs for cell-free expression, the use of reagents can be minimized by preparing small-scale cell-free reactions in a total inner volume of 50 µl and outer volume of 500 µl in the dialysis (Figure 2). Figure 2 illustrates the setup we use in our laboratory but other dialysis set ups can be used. For most proteins, a reaction volume of 50 µl should provide enough sample for running on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel to confirm the expression of the protein of interest and its relative yield across different reaction conditions.

    To produce larger quantities of proteins, the inner and outer volumes can be increased accordingly. Our large-scale production typically has an inner volume of 1 ml and an outer volume of 10 ml with each 1-ml reaction producing ~0.5-2 mg of a hydrophobin protein.

    To allow easy reproduction and scaling of the cell-free reactions, prepare the necessary reagents as combinations of mixtures as per Tables 1 and 2 and store at -80°C. The following protocol shows a small-scale cell-free reaction of 50 µl inner reaction volume and 500 µl outer reaction volume as described in Table 3. The concentration ranges for components that likely require optimization depending on the proteins of interest are given in Table 4. An interactive Excel spreadsheet that aids the calculation of reagents used in the reaction setup is available for download (Note 1).

Figure 2. Flowchart illustrating the setup of a small-scale cell-free reaction. The slashed lines indicate the position of the dialysis membrane.

  1. Preparation of reagent mixtures

    1. Prepare 3 mixtures of the 20 amino acids into 3 separate tubes on ice according to Steps A2 to A4 below.

    2. Add the amino acids (Ala, Arg, Gly, His, Lys, Pro, Ser, Thr, Val) and dissolve in MilliQ water to make water soluble amino acid stocks.

    3. Add the amino acids (Asn, Asp, Cys, Glu, Gln, Leu, Met, Trp, Tyr) and dissolve in 1 M HCl to make acid soluble amino acid stocks.

    4. Add the amino acids (Ile, Phe) and dissolve in 1 M KOH to make base soluble amino acid stocks.

    5. Mix the 3 components listed in Table 1 to make the total amino acid mixture with each amino acid at a final concentration of 15 mM.

      Table 1. Preparation of amino acids mixture for a 50-µl inner reaction

      Components Stock concentration Final concentration Volume
      Water soluble amino acid stock 50 mM 15 mM 12 µl
      Acid soluble amino acid stock 50 mM 15 mM 12 µl
      Base soluble amino acid stock 50 mM 15 mM 12 µl
      MilliQ water - - Fill up to 40 µl

    6. Mix all components listed in Table 2 on ice to prepare for the 10× pre-mix.

      Table 2. Preparation of 10× pre-mix for a 50 µl inner reaction

      Component Stock concentration Final concentration in reaction (1×) Final concentration (10×) Volume
      rNTP 25 mM 0.8 mM 8 mM 19.2 µl
      HEPES, pH 7.58 2 M 55 mM 550 mM 16.5 µl
      Folinic acid 10 mM 68 µM 680 µM 4.08 µl
      cAMP 100 mM 0.64 mM 6.4 mM 3.84 µl
      NH4OAc 10 M 27.5 mM 275 mM 1.65 µl
      MilliQ water - - - Fill up to 60 µl

    7. The amino acid mixtures and the 10× pre-mix can be stored at -80°C for later use.

  2. Preparation of inner and outer reaction master mixes

    1. Thaw the amino acid mix and the 10× pre-mix on ice.

    2. Add components of the inner reaction master mix into a new tube and outer reaction master mix into another tube (Table 3).

      Table 3. Preparation of inner and outer reaction master mix for a 50 µl inner reaction

      Component Stock concentration Final concentration in inner reaction Inner master mix (50 µl) Final concentration in outer reaction Outer master mix (500 µl)
      10× pre-mix 10× 5 µl 50 µl
      Creatine phosphate 1 M 80 mM 4 µl 80 mM 40 µl
      Amino acid mixture 15 mM 1 mM 3.3 µl 1 mM 33.33 µl
      Potassium Glutamate 4 M 208 mM 2.6 µl 208 mM 26 µl
      Creatine kinase 10 mg/ml 250 µg/ml 1.25 µl - -
      Mg(OAc)2 1 M 15 mM 0.75 µl 19.3 mM 9.65 µl
      ATP 100 mM 1.2 mM 0.6 µl 1.2 mM 6 µl
      tRNA 17.5 mg/ml 0.175 mg/ml 0.5 µl - -
      Total - - 18 µl 164.98 µl

    3. Keep the mixtures on ice while setting up the reaction apparatus.

  3. Cell-free reaction apparatus set-up

    1. Add components of inner reaction into a tube (Table 4).

      Table 4. Cell-free reaction set-up in a dialysis setting for a 50-µl inner reaction (use the provided Excel calculator and enter other volumes as required)

      Component Stock concentration Final concentration in inner reaction Inner Volume Final concentration in outer reaction Outer Volume
      Reaction master mix - - 18 µl - 164.65 µl
      S30 lysate - 30% (v/v) 15 µl - -
      DTTa 100 mM 1.7 mM 0.85 µl 1.7 mM 8.5 µl
      GSHb 10 mM 1 mM 5 µl 1 mM 50 µl
      GSSGc 5 mM 0.1 mM 1 µl 0.1 mM 10 µl
      DNAd 300 ng/µl 6 ng/µl 1 µl - -
      DsbCe 10 mg/ml 0.3 mg/ml 1.5 µl 0.3 mg/ml 15 µl
      MilliQ water Fill up to 50 µl Fill up to 500 µl

      aDTT (0.1-1.7 mM)

      bGSH (0.1-1 mM)

      cGSSG (0.1-0.1 mM)

      dDNA (0.3-6 ng/µl)

      eDsbC (0.1-1.7 mM)

      a−eCan be varied to meet the needs of the protein of interest

    2. Add components of outer reaction into another tube (Table 4).

    3. Adjust the outer reaction buffer to pH 7.5 before adding DTT, GSH, GSSG.

    4. Add Disulfide bond isomerase C (DsbC) (Note 2).

    5. Setup the apparatus for small-scale (Figure 3) or large-scale cell-free reaction.

      Figure 3. Preparation of a custom micro-dialysis button. A. Heat up a scalpel (e.g., using a Bunsen burner) and slice the tip of the tube and cut off the lid with scissors. B. Add inner reaction mixture to the lid, cover with a small piece of dialysis membrane. C. Seal the dialysis membrane onto the lid with the cut plastic tube, in the same orientation as per standard tube closure. Make sure the membrane is in place securely with excess dialysis membrane visible all around the lid. D. Add the outer reaction mixture to a collection tube that has a diameter slightly larger than the micro-dialysis button. Place the micro-dialysis button inside this tube.

    6. For a small-scale reaction, make a micro-dialysis button by slicing the lid of a 0.45-ml PCR tube.

    7. Place the lid upside down on a flat surface and add 50 μl of the inner reaction mixture (or any other desirable volume that fits) to the lid.

    8. Wet a small stretch of 10,000 MWCO SnakeSkin® dialysis membrane with water, dab off the excess water with a tissue paper and wedge the membrane between the lid and the cut tube.

    9. Add the outer reaction mixture to a tube and place the micro-dialysis button inside with the membrane exposed to the outer reaction mixture.

    10. Incubate the cell-free reaction for 4-20 h at 30°C and 180 rpm by taping it to a holder in a shaking incubator.

  4. Initial analysis of protein expression

    1. Once the reaction has been completed, remove the dialysis buttons and dab dry. Then carefully pierce the membrane with a pipette tip with pipette plunger depressed to the first stop and aspirate the inner reaction mixture for downstream analysis.

    2. Clarify the reaction mixture by centrifugation (20,000 × g, 10 min, 4°C) and separate soluble and insoluble fractions and aliquot appropriate amounts for SDS-PAGE analysis.

    3. For 20-µl SDS-PAGE gel samples, take 10 µl of the clarified soluble fraction and add 10 µl of 1× PBS, and take the insoluble fraction and dilute with 20 µl of 1 M Urea.

    4. Add 1× LDS buffer from 4× LDS stock to the gel samples and heat for 10 min at 80°C.

    5. Run SDS-PAGE analysis at 200 V for 20 min and stain according to manufacturer’s protocol.

  5. Purification of the expressed protein

    This will vary depending on the protein of interest and whether an affinity tag is present. For the hydrophobin proteins produced in our laboratory, we use Nickel-NTA Affinity Chromatography (IMAC), followed by cleavage and removal of the hexa-histidine tag when needed and then reverse-phased High Pressure Liquid Chromatography (rpHPLC). The use of isotopically labeled amino acids (e.g., with 15N) can circumvent the need for protein purification in certain applications (e.g., to check protein conformation using 1H-15N NMR spectroscopy) which can lead to a substantial time saving.

  6. Analysis of the purified protein

    The analysis of the purified protein, including measuring its yield will vary according to the specific characteristics of the protein of interest as well as the available equipment. Several techniques such as gel densitometry, Bradford assays or UV-Vis spectroscopy can be utilized to measure the yield of the purified protein. An activity assay (e.g., enzymatic activity assay) for the protein of interest should be conducted if available to assess if the produced protein is functional. With our proteins of interest, we check protein purity using SDS-PAGE, calculate the yield of the hydrophobin proteins by measuring absorbance of the purified protein solution at 280 nm using a NanoDropTM, confirm the correct folding via NMR spectroscopy, confirm the mass of the protein matches that of the calculated mass via mass spectrometry and validate its ability to assemble using Thioflavin-T assays.


  1. An interactive cell-free reaction set-up calculator is available to download. On the left-hand side, the reaction is shown as a small-scale cell-free reaction with a 50-μl inner reaction volume. These values are used on the right-hand side within the reaction set-up calculator. Any desired inner reaction volume can be entered along with the number of reactions and the required volumes will be calculated. The desired concentrations of the redox additives (DTT, GSH:GSSG, DsbC) and the concentration of DNA can be adjusted as required (see Table 4 for suggested ranges).

  2. The DsbC was expressed and purified as described in Matsuda et al. (2013) and dialyzed into Phosphate-buffered saline (PBS) and stored at 4°C until use. The construct we used was obtained from the Kigawa laboratory.

  3. It is important to choose a plasmid that allows for constitutive protein expression, i.e., without the lac operator. The plasmid we used is the pET-MCSIII as this vector has been reported for the successful synthesis of a number of proteins in cell free synthesis schemes. However, we believe that any recombinant protein expression vector can be used with the protocol provided constitutive protein expression can be achieved. If an existing recombinant protein expression vector has a lac operator, we suggest removing this to increase the yield. Instead of plasmid DNA, the use of PCR-amplified DNA can also be used with this set up (Neylon et al., 2000; Wu et al., 2007).

  4. If production of proteins with selectively labeled amino acid(s) is required, simply replace the unlabeled amino acid(s) used in Steps A2, A3 and/or A4 with the labeled amino acid(s) of choice when making up the amino acid mixture (Table 1).


This project was supported by a Discovery Project grant, DP200102463 from the Australian Research Council to A.H.K.

Competing interests

The authors declare no competing interests.


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  2. Ball, S. R., Pham, C. L. L., Lo, V., Morris, V. K., Kwan, A. H. and Sunde, M. (2020). Formation of Amphipathic Amyloid Monolayers from Fungal Hydrophobin Proteins. Methods Mol Biol 2073: 55-72.
  3. Berger, B. W. and Sallada, N. D. (2019). Hydrophobins: multifunctional biosurfactants for interface engineering. J Biol Eng 13: 10.
  4. Berkmen, M. (2012). Production of disulfide-bonded proteins in Escherichia coli. Protein Expr Purif 82(1): 240-251.
  5. Dondapati, S. K., Stech, M., Zemella, A. and Kubick, S. (2020). Cell-Free Protein Synthesis: A Promising Option for Future Drug Development. BioDrugs 34(3): 327-348.
  6. Gao, W., Cho, E., Liu, Y. and Lu, Y. (2019). Advances and Challenges in Cell-Free Incorporation of Unnatural Amino Acids Into Proteins. Front Pharmacol 10: 611.
  7. Kadokura, H. and Beckwith, J. (2010). Mechanisms of oxidative protein folding in the bacterial cell envelope. Antioxid Redox Signal 13(8): 1231-1246.
  8. Khambhati, K., Bhattacharjee, G., Gohil, N., Braddick, D., Kulkarni, V. and Singh, V. (2019). Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems. Front Bioeng Biotechnol 7: 248.
  9. Kwan, A. H., Winefield, R. D., Sunde, M., Matthews, J. M., Haverkamp, R. G., Templeton, M. D. and Mackay, J. P. (2006). Structural basis for rodlet assembly in fungal hydrophobins. Proc Natl Acad Sci U S A 103(10): 3621-3626.
  10. Matsuda, T., Watanabe, S. and Kigawa, T. (2013). Cell-free synthesis system suitable for disulfide-containing proteins. Biochem Biophys Res Commun 431(2): 296-301.
  11. Neylon, C., Brown, S. E., Kralicek, A. V., Miles, C. S., Love, C. A. and Dixon, N. E. (2000). Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry 39(39): 11989-11999.
  12. Ozawa, K. and Loh, C. T. (2014). Site-specific incorporation of unnatural amino acids into proteins by cell-free protein synthesis.Methods Mol Biol 1118: 189-203.
  13. Pham, C. L., Rey, A., Lo, V., Soules, M., Ren, Q., Meisl, G., Knowles, T. P., Kwan, A. H. and Sunde, M. (2016). Self-assembly of MPG1, a hydrophobin protein from the rice blast fungus that forms functional amyloid coatings, occurs by a surface-driven mechanism. Sci Rep 6: 25288.
  14. Pille, A., Kwan, A. H., Cheung, I., Hampsey, M., Aimanianda, V., Delepierre, M., Latge, J. P., Sunde, M. and Guijarro, J. I. (2015). 1H, 13C and 15N resonance assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus. Biomol NMR Assign 9(1): 113-118.
  15. Qin, M., Wang, W. and Thirumalai, D. (2015). Protein folding guides disulfide bond formation. Proc Natl Acad Sci U S A 112(36): 11241-11246.
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图1.使用亲和色谱法进行无细胞蛋白质表达和一步纯化的工作流程。一。如Apponyi等人所述制备大肠杆菌S30裂解物。(2008 )可以在-80的温度下存储长达几年 °C,根据我们的经验,没有任何活动损失。乙。在S30裂解物,质粒DNA,对与亲和标签沿着感兴趣的蛋白进行编码和转录和翻译必需的组件加编到反应混合物中。一步纯化蛋白质后,可以分离目标蛋白质以进一步使用。


常规重组细菌过表达的一种替代方法是无细胞合成,其中可以使用细菌转录和蛋白质生产机制表达蛋白质,但没有细胞的限制(Apponyi等,2008 ; Ozawa和Loh,2014 ; Gao等。等,2019;Dondapati,2020 )。无细胞方案对于表达富含半胱氨酸的蛋白质特别有用,因为可以通过简单地改变还原型和氧化型谷胱甘肽(GSH:GSSG)的比例以及改变还原剂(如二硫苏糖醇(DTT))的浓度来控制氧化还原环境。)。后优化,以正确地形成蛋白质的产量二硫化物键适于下游实验(例如,结合研究,结构和生物物理表征)可以大大增加。

除具有二硫键的蛋白质外,无细胞蛋白质合成系统与传统的重组蛋白质生产相比,还具有其他优势,例如能够轻易掺入非天然氨基酸(Gao等人,2019 ;Dondapati等人,2020)。此外,太毒了宿主细胞蛋白可以在无细胞环境中表达,因为没有需求来维持细胞活力,这也使得高通量生产和筛选膜蛋白,酶和其他疗法 (Khambhati et al。,2019)。

我们优化的无细胞表达方案允许直接控制反应混合物中的氧化还原条件,并显示出优于重组表达的一类称为真菌疏水蛋白的蛋白质的明显优势(Siddiquee等,2020)。这类蛋白质的特征在于四个二硫键,疏水蛋白在亲水:疏水界面自组装成两亲性层需要正确的键合模式(Sunde等,2008 ;Ball等,2020)。第ESE独特assembl IES公顷VE建议的范围内的药物递送系统的生物技术应用到涂覆植入物和所述表面中的工程以提高生物相容性(Wosten和Scholtmeijer,2015 ;伯杰和Sallada,2019) 。W¯¯往往微不足道许多疏水蛋白可以在过表达大肠杆菌到一个非常高的收益率,蛋白质几乎只发现错误折叠的包涵体内,而不能用于下游应用。因此,通常需要很长的体外重折叠和多步纯化过程才能从部分折叠和错误折叠的物种中分离正确折叠的蛋白质(Kwan等人,2006; Pille等人,2015 ;Pham等人, 2016)。为了克服这个问题,我们已经制定了一个无细胞表达系统疏水蛋白的生产基础上的发布S30大肠杆菌裂解液(Apponyi等,2008) 。

S30裂解物是无细胞蛋白质合成中最常用的提取物之一,含有细胞转录和翻译机制。使用“ S30无细胞”作为搜索词的Pubmed搜索可得到150篇以上的文章。虽然S30裂解物可以商购(例如,来自Promega),它可以容易地制造在执行重组蛋白质表达和纯化实验室在成本的一小部分的可商购的无细胞表达的试剂盒。此无细胞反应包括温育裂解物和其它关键辅因子与所关注的基因克隆到透析秒的组成型启动子的下游,等起来。设置透析允许较低的分子的试剂(发生连续交换例如,氨基酸,的rNTPs ,ATP),并提供每单位更高的蛋白产量组分是更昂贵或费时的来制备(例如,在转录和翻译机器S30提取物以及质粒DNA )。

为了帮助纯化,我们将该基因克隆到了具有六组氨酸亲和标签但缺乏lac操纵子的载体pET -MCSIII中(Neylon等,2000 )。本文介绍的方案可以轻松修改,以表达具有二硫键的其他蛋白质。该协议中使用的设备可以使用大多数科学实验室中常用的一次性塑料管轻松制备,并且可以根据需要进行放大。的效率和低成本这组向上允许多个蛋白质(例如,蛋白质和其突变体)以被并行无细胞反应使用一种生产制备的透析缓冲液中。同位素标记的蛋白还可以容易地通过替代衔接产生纳克在标记的氨基酸在该反应混合物中。我Ñ一些情况下小号,这已经允许核磁共振(NMR)和质谱实验的目的蛋白上的一天内进行设定的反应而不需要进一步纯化。

关键字:疏水蛋白, 二硫键, 蛋白质合成, 无细胞的, 蛋白质生产, DsbC


1.的Eppendorf吨ubes1.5毫升(热Fisher Scientific公司,目录号:69715)     


3. Greiner Bio-One 50毫升试管     



6. rNTP (Sigma-Aldrich,目录号:A6559,C8552,G3776,U1006)     

7. HEPES(Sigma-Aldrich,目录号:15630106)     


9. cAMP(Sigma-Aldrich,目录号:A6885)     

10. NH 4 OAc(西格玛奥德里奇,目录号:A1542) 

11. ATP(西格玛奥德里奇,目录号:A1852) 

12.肌酸p hosphate(Sigma-Aldrich公司,目录号:27920) 


14.肌酸ķ inase(Sigma-Aldrich公司,目录号:C9983) 

15. Mg(OAc )2 (Sigma-Aldrich,目录号:M5661) 

16. tRNA(西格玛奥德里奇,目录号:R8759) 



19. L-谷胱甘肽oxidis ED (GSSG)(Sigma-Aldrich公司,目录号:G4376) 

20. 20个氨基酸(Sigma-Aldrich) 

21.万MWCO蛇皮®透析膜(热Fisher Scientific公司,目录号:88243)。请注意,选择的膜截面积至少要比预期的蛋白质产品小三倍 

22. NuPAGE TM 4-12%Bis-Tris蛋白凝胶(Invitrogen ,目录号:NP0323PK2 )或任何适用于检测目标蛋白的SDS-PAGE凝胶 

23. MES缓冲液(Invitrogen ,目录考勤号:NP0002)或其它相容的缓冲液为运行SDS-PAGE凝胶 

24. NuPAGE TM LDS样品缓冲液(4 × )(Thermo Fisher Scientific,目录号:NP0007) 

25. PBS(Sigma-Aldrich,目录号:P4417) 


27.重组二硫键异构酶DsbC (注2) 

28.质粒或PC ř -a米p升ifie d FR agmen吨编码感兴趣的蛋白(注3) 



NanoDrop TM紫外可见分光光度计(Thermo Fisher Scientific,目录号:ND-2000 )
保温箱(Eppendorf ,型号:Innova 44 ;M1282-0002)
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS- P AGE)装置(赛默飞世科学,目录号:A25977 )






当测试新构建体的无细胞表达时,可以通过在透析中以总内部体积为50μl,外部体积为500μl进行小规模的无细胞反应来最大程度地减少试剂的使用(图2)。图2说明了我们在我们的实验室,但其他透析组使用了设置小号都可以使用。对于大多数蛋白质,反应体积为50 µl,应提供足够的样品,以便在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE )凝胶上运行,以确认目标蛋白在不同反应条件下的表达及其相对产率。

为了产生更大量的蛋白质,所述内和外体积S可根据增加LY 。我们的大规模生产通常具有的内部容积1毫升和一个外部体积的10毫升与每个1毫升反应产生〜0.5 - 2毫克一个的疏水蛋白。

为了使无细胞反应易于复制和缩放,请按照表1和表2的混合物准备必要的试剂,并在-80°C下保存。以下方案显示了表3中所述的内部反应体积为50 µl和外部反应体积为500 µl的小规模无细胞反应。表4给出了可能需要根据目标蛋白质进行优化的组分的浓度范围。可下载交互式Excel电子表格,该电子表格可帮助计算反应设置中使用的试剂(注1)。



根据以下步骤A 2至A 4 ,将20种氨基酸的3种混合物准备在冰上的3个单独试管中。
添加的氨基酸(异亮氨酸,苯丙氨酸)和在1M KOH溶解,使碱溶性氨基酸股票小号。
混合表1中列出的3种成分,使总氨基酸混合物与每种氨基酸的最终浓度为15 mM。

表1.用于50 µl内部反应的氨基酸混合物的制备




















最多填充40 µl

混合所有成分中列出。表2在冰上制备用于10 ×预混合。

表2.用于50 µl内部反应的10 ×预混合物的制备



反应终浓度(1 × )

最终浓度(10 × )







pH 7.58的HEPES

2 M













NH 4 OAc

10 M








最多填充60 µl

氨基酸混合物和10 ×预混合物可以在-80°C下保存以备后用。

解冻的氨基酸混合物和所述10 ×在冰上预混合。

表3.用于50 µl内部反应的内部和外部反应预混合物的制备




内部预混液(50 µl )


外部预混液(500 µl )

10 ×预混

10 ×

1 ×


1 ×



1 M























镁(OAc )2

1 M
























表4.用于50 µl内部反应的透析设置中的无细胞反应设置(使用提供的Excel计算器并根据需要输入其他体积)















30%(v / v)























300 ng /微升

6 ng /微升











最多填充50 µl

填充量最大为500 µl

一个DTT(0.1 - 1.7毫摩尔)           

b GSH (0.1 - 1毫米)

Ç GSSG (0.1 - 0.1毫米)

d DNA(0.3 - 6纳克/微升)           

È的DsbC (0.1 - 1.7毫米)

A- ê可以被改变以满足目标蛋白质的需求

在添加DTT,GSH,GSSG之前,将外部反应缓冲液的pH调节至7.5 。
加入二硫键异构酶C (DsbC )(注2)。

图3.准备一个自定义的微透析按钮。一。加热手术刀(例如,使用本生灯),将试管的尖端切成薄片,并用剪刀剪下盖子。乙。将内部反应混合物加到盖子上,盖上一小块透析膜。Ç 。用切开的塑料管将透析膜密封在盖子上,其方向与标准管的闭合方向相同。确保膜是在地方安全地与周围的盖多余的透析膜中所有可见。d 。将外部反应混合物添加到直径略大于微透析按钮的收集管中。将微透析按钮放在该管内。

对于小规模的反应,请切成0.45 ml PCR管的盖子,制成微透析按钮。
放置在平坦的表面上的盖子倒置和DD 50 μ升的的内反应混合物(或任何其它期望的体积适合)到盖子。

一旦反应已经完成,REM OVE透析按钮和DAB干。然后用移液器尖端小心地刺穿膜,将移液器柱塞压下至第一个停止点,然后吸出内部反应混合物以进行下游分析。
通过离心(20,000 ×g ,10分钟,4°C)澄清反应混合物,分离可溶性和不溶性馏分,并分装适当量的SDS-PAGE分析。
对于20 µl SDS-PAGE凝胶样品,取10 µl澄清的可溶级分,加入10 µl 1 × PBS,取不溶级分,并用20 µl 1 M尿素稀释。
从4 × LDS储备液中添加1 × LDS缓冲液到凝胶样品中,并在80°C加热10分钟。
在200 V下运行SDS-PAGE分析20分钟,并根据制造商的规程染色。

这将根据目标蛋白质以及是否存在亲和标签而有所不同。对于实验室中产生的疏水蛋白,我们使用镍-NTA亲和色谱(IMAC),然后在需要时裂解并去除六组氨酸标签,然后反相高压液相色谱(rpHPLC )。标记的氨基酸(例如,用15 N)可以规避在某些应用蛋白质纯化的需要(例如,以检查使用蛋白质构象1 H- 15 ñNMR光谱法),其可导致大量的时间节省。

纯化蛋白质的分析(包括测量其产率)将根据目标蛋白质的具体特征以及可用的设备而有所不同。几种技术如凝胶光密度,布拉德福德分析或UV-Vis光谱,可以利用来测量纯化的蛋白质的产率。一个一个ctivity测定法(例如,酶活性测定)对于感兴趣的蛋白质应该进行(如果可用)来评估,如果所产生的蛋白质是功能性的。使用我们感兴趣的蛋白质,我们使用SDS-PAGE检查蛋白质的纯度,通过使用NanoDrop TM测量纯化的蛋白质溶液在280 nm处的吸光度来计算疏水蛋白的收率,通过NMR光谱确认正确的折叠,确认该蛋白质可通过质谱法与计算质量相匹配,并使用硫黄素-T分析验证其组装能力。


可以下载交互式无细胞反应设置计算器。在左侧,该反应显示为小规模的无细胞反应,其中50-μ升内体积的反应。这些值在反应设置计算器的右侧使用。任何期望的内反应体积可以一起输入的反应的数量和所需的v olumes将被计算。氧化还原添加剂(DTT,GSH的所需的浓度:GSSG ,DsbC的)和DNA的浓度可根据需要进行调整(见表4为表明编范围)。
如Matsuda等所述,表达和纯化DsbC 。(2013 )并透析入磷酸盐缓冲盐水(PBS)中,并储存在4°C直至使用。我们使用的构建体是从Kigawa实验室获得的。
选择一个质粒是非常重要的是所有Ø WS的利弊牛逼它utiv é亲TEIN é XPR é SSIO ñ ,即,没有lac操纵。我们用质粒是吨他的pET -MCSIII作为本VECT ö ř ħ作为被报道为成功小号一些蛋白质的ynthesis在无细胞合成方案。但是,我们相信,只要可以实现组成性蛋白表达,任何重组蛋白表达载体均可与该方案一起使用。如果现有的重组蛋白表达载体具有lac操纵子,我们建议将其删除以提高产量。代替质粒DNA,也可以在该设置中使用PCR扩增的DNA(Neylon等,2000 ;Wu等,2007 )。
如果需要生产带有选择性标记氨基酸的蛋白质,只需在选择氨基酸时将步骤A2,A 3和/或A 4中使用的未标记氨基酸替换为所选的标记氨基酸即可。氨基酸混合物(表1)。






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引用:Siddiquee, R. and Kwan, A. H. (2021). Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins. Bio-protocol 11(10): e4019. DOI: 10.21769/BioProtoc.4019.

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