Materials and Reagents

Eppendorf tubes 1.5 ml (Thermo Fisher Scientific, catalog number: 69715)
Eppendorf tubes 0.5 ml (Eppendorf, catalog number: 0030121023)
Greiner Bio-One 50 ml tubes
Pipette tips
S30 Lysate prepared according to Apponyi et al. (2008)
rNTP (Sigma-Aldrich, catalog numbers: A6559, C8552, G3776, U1006)
HEPES (Sigma-Aldrich, catalog number: 15630106)
Folinic acid (Sigma-Aldrich, catalog number: 47612)
cAMP (Sigma-Aldrich, catalog number: A6885)
NH₄OAc (Sigma-Aldrich, catalog number: A1542)
ATP (Sigma-Aldrich, catalog number: A1852)
Creatine phosphate (Sigma-Aldrich, catalog number: 27920)
Potassium glutamate (Sigma-Aldrich, catalog number: G1501)
Creatine kinase (Sigma-Aldrich, catalog number: C9983)
Mg(OAc)₂ (Sigma-Aldrich, catalog number: M5661)
tRNA (Sigma-Aldrich, catalog number: R8759)
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)
L-Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251)
L-Glutathione oxidised (GSSG) (Sigma-Aldrich, catalog number: G4376)
20 amino acids (Sigma-Aldrich)
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
NuPAGE^TM 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
MES buffer (Invitrogen, catalog number: NP0002) or other compatible buffers for the running the SDS-PAGE gel
NuPAGE^TM LDS Sample Buffer (4×) (Thermo Fisher Scientific, catalog number: NP0007)
PBS (Sigma-Aldrich, catalog number: P4417)
Urea (Sigma-Aldrich, catalog number: U5378)
Recombinant disulphide isomerase DsbC (Note 2)
Plasmid or PCR-amplified fragment encoding for the protein of interest (Note 3)

Equipment

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.

Scalpel
NanoDrop^TM UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: ND-2000)
Bunsen burner
Incubator (Eppendorf, model: Innova 44; M1282-0002)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus (ThermoFisher Scientific, catalog number: A25977)

Software

Microsoft Excel

Procedure

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.

Preparation of reagent mixtures

Prepare 3 mixtures of the 20 amino acids into 3 separate tubes on ice according to Steps A2 to A4 below.
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.
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.
Add the amino acids (Ile, Phe) and dissolve in 1 M KOH to make base soluble amino acid stocks.

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

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

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

Preparation of inner and outer reaction master mixes

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

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×	1×	5 µl	1×	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

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

Cell-free reaction apparatus set-up

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	-	-
DTT^a	100 mM	1.7 mM	0.85 µl	1.7 mM	8.5 µl
GSH^b	10 mM	1 mM	5 µl	1 mM	50 µl
GSSG^c	5 mM	0.1 mM	1 µl	0.1 mM	10 µl
DNA^d	300 ng/µl	6 ng/µl	1 µl	-	-
DsbC^e	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

Add components of outer reaction into another tube (Table 4).
Adjust the outer reaction buffer to pH 7.5 before adding DTT, GSH, GSSG.
Add Disulfide bond isomerase C (DsbC) (Note 2).
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.
For a small-scale reaction, make a micro-dialysis button by slicing the lid of a 0.45-ml PCR tube.
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.
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.
Add the outer reaction mixture to a tube and place the micro-dialysis button inside with the membrane exposed to the outer reaction mixture.
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.

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.
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.
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 NanoDrop^TM, 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.

Notes

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).
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.
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).
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).

Acknowledgments

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.

References

Apponyi, M. A., Ozawa, K., Dixon, N. E. and Otting, G. (2008). Cell-free protein synthesis for analysis by NMR spectroscopy. Methods Mol Biol 426: 257-268.
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.
Berger, B. W. and Sallada, N. D. (2019). Hydrophobins: multifunctional biosurfactants for interface engineering. J Biol Eng 13: 10.
Berkmen, M. (2012). Production of disulfide-bonded proteins in Escherichia coli. Protein Expr Purif 82(1): 240-251.
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.
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.
Kadokura, H. and Beckwith, J. (2010). Mechanisms of oxidative protein folding in the bacterial cell envelope. Antioxid Redox Signal 13(8): 1231-1246.
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.
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.
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.
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.
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.
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.
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.
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.
Siddiquee, R., Choi, S. S., Lam, S. S., Wang, P., Qi, R., Otting, G., Sunde, M. and Kwan, A. H. (2020). Cell-free expression of natively folded hydrophobins. Protein Expr Purif 170: 105591.
Sunde, M., Kwan, A. H., Templeton, M. D., Beever, R. E. and Mackay, J. P. (2008). Structural analysis of hydrophobins. Micron 39(7): 773-784.
Wosten, H. A. and Scholtmeijer, K. (2015). Applications of hydrophobins: current state and perspectives. Appl Microbiol Biotechnol 99(4): 1587-1597.
Wu, P. S., Ozawa, K., Lim, S. P., Vasudevan, S. G., Dixon, N. E. and Otting, G. (2007). Cell-free transcription/translation from PCR-amplified DNA for high-throughput NMR studies. Angew Chem Int Ed Engl 46(18): 3356-3358.