Refolding by Dialysis

1 M urea and sonicated four times for 50 s at 70% of the maximum power in an ultrasonic homogenizer 4710 (Cole-Parmer Instrument Co.; Chicago, IL, USA), followed by centrifugation (27,000g for 40 min at 4 °C). The inclusion body wash step was repeated three times. Subsequently, the material collected by centrifugation after the last washing step was resuspended in 10 mL of buffer A. The total washed inclusion bodies were divided into two 100 mL beakers and shaken on an orbital shaker at 30 rpm at room temperature. Each fraction of the washed inclusion bodies was denatured by adding a freshly prepared 8 M urea solution dropwise with the aid of a Pasteur pipette until a transparent and homogeneous solution was obtained (approximately 40 mL of 8 M urea was added to each beaker). The denatured protein samples were initially dialyzed at a 1:10 ratio against buffer A containing 10% v/v glycerol and 0.1 mM EDTA (refolding buffer) at 4 °C for 4 h. Afterward, another dialysis step was performed at a 1:100 ratio against the same buffer at 4 °C for 16 h. The solubilized protein sample was recovered by centrifugation (27,000g for 40 min at 4 °C). The XfPal protein purification was performed in a single gravity flow chromatography step using a column packed with 1 mL of Ni–NTA resin (Qiagen; Germany) equilibrated with buffer A. The purified XfPal protein was eluted using a three-step gradient of imidazole (100, 250 and 500 mM) in buffer A, each step containing 7 mL of the respective buffer. The purity of the protein samples was estimated by SDS–PAGE and staining with Coomassie brilliant blue R-250. The total protein concentration was determined using a micro BCA™ protein assay kit or by spectroscopy using a calculated molar absorption coefficient at 280 nm (ε280) of 16,180 M−1/cm for the purified protein sample.

1. Isolation of inclusion bodies

Inclusion bodies have a relatively high density and, therefore, can be pelleted by centrifugation. Cells are usually disrupted by high pressure homogenization (optionally following a lysosyme treatment). It is important that cell lysis is complete, because intact cells sediment together with the inclusion bodies, thus contaminating the preparation. After centrifugation, the pellet is washed with buffer containing either low concentrations of chaotropic agents (e.g.0.5-1 M guanidine-HCl or urea) or detergents (e.g. 1% Triton X-100 or 1 mg/ml sodium deoxycholate). This wash step is necessary to remove contaminants, especially proteins (proteases), that may have absorbed onto the hydrophobic inclusion bodies during processing.

2. Solubilization of aggregated proteins

The washed inclusion bodies are resuspended and incubated in buffer containing a strong denaturant and a reducing agent (usually 20 mM DTT or b-mercaptoethanol). The addition of a reducing agent keeps all cysteines in the reduced state and cleaves disulfide bonds formed during the preparation. Incubation temperatures above 30°C are typically used to facilitate the solubilization process. Optimal conditions for solubilization are protein specific and have to be determined for each protein. This is done most effectively by carrying out small-scale experiments (1-2 ml) to screen the different variables.

Important variables for solubilization are:

Variable good starting point buffer composition (pH, ionic strength) 50 mM Tris-HCl, pH 7.5 incubation temperature 30°C incubation time 60 min concentration of solubilzing agent 6 M guanidine-HCl or 8 M urea total protein concentration 1-2 mg/ml ratio of solubilizing agent to protein After solubilization the solution should be centrifuged to remove remaining aggregates which could act as nuclei to trigger aggregation during refolding. The best results are obtained when we use ultracentrifugation (30 min at >100,000 g).

3. Refolding of the solubilized proteins

Refolding of the solubilized proteins is initiated by the removal of the denaturant. The efficiency of refolding depends on the competition between correct folding and aggregation. To slow down the aggreagtion process refolding is usually carried out at low protein concentrations, in the range of 10-100 mg/ml. Furthermore, refolding conditions must be optimized for each individual protein. Important variables are:

buffer composition (pH, ionic strength) temperature additives (often in combination)

FoldIt, a commercial protein folding screen is available from Hampton Research. If proteins contain disulfide bonds, the refolding buffer has to be supplemented with a redox system. The addition of a mixture of reduced and oxidized forms (1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol) of low molecular weight thiol reagent usually provides the appropriate redox potential to allow formation and reshuffling of disulfide bonds. The most commonly used redox shuffling reagents are reduced and oxidized glutathione, but also cysteine and cysteamine are used. For certain protein, probably due to low solubility of folding intermediates, this procedure is not very effective. Alternatively, the protein is completely oxidized in the presence of a large excess of oxidized glutathione, followed by dilution in refolding buffer containing catalytic amounts of reduced glutathione.

Different methods for the refolding of proteins have been described:

Dialysis.

The most used method is the removal of the solubilizing agent by dialysis. During dialysis the concentration of the solubilizing agent decreases slowly which allows the protein to refold optimally. The ratio of the volumes of the sample and the dialysis buffer should be as such that at the equilibrium concentration of the solubilizing agent the protein has completely refolded.

Slow dilution.

The concentration of the solubilizing agent is decreased by dilution allowing the protein to refold. Usually the dilution is carried out slowly by step-wise addition of buffer or by continuous addition using a pump.

Rapid dilution. During dialysis and slow dilution the protein is exposed for an extended period of time to an intermediate concentration of the solubilizing agent (2-4 M urea or guanidine-HCl) where it is not yet folded but no longer denatured and thus extremely prone to aggregation. This could be prevented by the rapidly dilution of the solubilized protein solution into the refolding buffer. Aggregation during this process can be limited by adding mild solubilizing agents to the refolding buffer, such as non-detergent sulfobetaines.

Pulse renaturation. In order to keep the concentration of the unfolded protein low, thus limiting aggregation, aliquots of denatured protein are added at defined time points to the refolding buffer. The time intervals between two pulses have to be optimized for each individual protein.The process is stopped when the concentration of denaturant reaches a critical level with respect to refolding of the specific protein.

Chromatography. The solubilizing agent is removed using a chromatographic step. The application of different chromatography methods have been described:

size exclusion chromatography (e.g. gel filtration on a Superdex 75 column) ion exchange chromatography affinity chromatography (e.g. IMAC using Chelating Sepharose or Ni-NTA agarose) The denaturant is removed while the protein is slow migrating through the column or bound to the matrix. This usually gives a high yield of active protein even at protein concentrations in the mg/ml range. Alternatively, it is possible to carry out chromatography under denaturing conditions before refolding the protein. Most modern chromatography resins are stable under the commonly used conditions for solubilization.

GM-CSF purification https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049891

Isolation of Inclusion Bodies Cells were resuspended in 25 ml of lysis buffer (50 mM Tris, pH 8, 2 mM EDTA, 0.5% Triton X-100 with the addition of Complete Mini Protease Inhibitor Tablets (Roche)). Mechanical disruption was used to lyse the cells (i.e. French Press or sonication) and the inclusion bodies isolated from the cell supernatant by centrifugation at 18000×g at 2–8°C for 20 minutes. The inclusion body pellet was solubilized in resuspension buffer (50 mM Tris, pH 8, 6 M GuHCl (Sigma, G4505), 10 mM DTT) by repeatedly passing the inclusion bodies through an 18g syringe. It is worth noting that any insoluble material can be centrifuged out at this time at 18000×g at 2–8°C for 20 minutes. The resuspended protein material was then diluted 50% in dialysis buffer #1 (50 mM Tris, pH 8, 2 M GuHCl) resulting in a 4 M GuHCl containing solution. The protein solution was then dialyzed overnight at 4°C in snakeskin dialysis tubing (Pierce) against 2 L of buffer #1. The following day the dialysis buffer was changed to 2 L of dialysis buffer #2 (50 mM Tris, pH 8, 1 M GuHCl, 0.4 M Arginine (Sigma, A5006), 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione) for overnight dialysis at 4°C. The following day the dialysis buffer was diluted 50% with water and dialysis continued overnight. Any insoluble material was centrifuged (18000×g at 2–8°C for 20 minutes) and the remaining protein solution dialyzed overnight at 4°C against 1 L of dialysis buffer #3 (50 mM Tris, pH 8, 250 mM NaCl, 0.1 M Arginine, 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione) to remove the remaining GuHCl.

rhGM-CSF Purification The final dialyzed protein solution was clarified by centrifugation (18000×g at 2–8°C for 20 minutes) and the supernatant loaded onto a ProBond (Invitrogen) His-tag affinity column equilibrated in equilibration buffer (50 mM Tris, pH 8, 500 mM NaCl). The column was washed with an excess of wash buffer (50 mM Tris, pH 8, 1 M NaCl, 0.1% Triton X-100) followed by a wash with equilibration buffer to remove the Triton X-100. The rhGM-CSF was eluted in 1 ml aliquots from the resin using elution buffer (50 mM Tris, pH 8, 250 mM Immidazole). To quickly visualize the eluted protein, 10 µl from individual fractions was pipetted on Whatmann filter paper, and stained with Coomassie Brilliant Blue for 2 min before destaining or analyzed by 15% reducing SDS PAGE. Fractions containing eluted protein were pooled and dialyzed against 2 L of 20 mM HEPES, pH 7.8, overnight at 4°C. Protein that precipitated out during the overnight low salt dialysis was removed by centrifugation before proceeding. The purified rhGM-CSF was quantified by UV spectroscopy using a calculated molar absorption coefficient corresponding to the non-reduced rhGM-CSF sequence (e280nm = 14 238) [20]. With the additional LEHHHHHHHH at the C-terminus and resulting higher molecular weight, the absorbance value (A0.1%) of the folded rhGM-CSF at 280 nm is 0.89 compared to 0.98 of rhGM-CSF minus the additional amino acids.

Inclusion Bodies Purification Protocol

In many cases, high-level expression of recombinant proteins leads to the formation of protein aggregation commonly called as inclusion bodies. In order to obtain biologically active and soluble protein in high yield, inclusion bodies must then be solubilized and refolded in vitro.The first step is purification of inclusion bodies. For more information about protein refolding, you can refer to Overview of Protein Refolding.

Isolation and purification Inclusion bodies can be pelleted by centrifugation for a relatively high density and cells are usually disrupted by high pressure homogenization (optionally following a lysosyme treatment). It is important that cell lysis is complete, because intact cells sediment together with the inclusion bodies, thus contaminating the preparation. After centrifugation, the pellet is washed with buffer containing either low concentrations of chaotropic agents (e.g. 0.5-1 M guanidine-HCl or urea) or detergents (e.g. 1% Triton X-100). This wash step is necessary to remove contaminants, especially proteins (proteases), that may have absorbed onto the hydrophobic inclusion bodies during processing.

Solubilization of inclusion bodies protein The washed inclusion bodies are resuspended and incubated in buffer containing a strong denaturant and a reducing agent (usually 20 mM DTT or β-mercaptoethanol). The addition of a reducing agent keeps all cysteines in the reduced state and cleaves disulfide bonds formed during the preparation. Optimal conditions for solubilization are protein specific and have to be determined for each protein.

Purification

Spin down cells from large culture at 6000 rpm for 20 min.

Suspend the cell pellet (from 1L culture) in 30-35ml of PBST buffer. To facilitate lysis and inclusion body purification, add 0.5–1.0 % Triton X-100. Some EDTA and DTT, up to 50 mM, should be used in all subsequent steps to keep disulfides reduced. The cells can be lysed with either French press or sonication. With French press the cells should be passed at least twice through to ensure complete shearing of the genomic DNA. Sonication conditions should be optimised depending on the sonicator, tip, amount of cells in per volume of lysis buffer etc.

Centrifuge cell lysate for approximately 20 min at 15,000 rpm, 4°C.

Discard the supernatant.

Wash the inclusion body pellets in a small volume of buffer containing 1-2% Triton X-100. This should solubilise membranes and membrane proteins. A short sonication (3 x 10 seconds) is very helpful during each wash step. Not only will it help to resuspend all the inclusion bodies, but it will also break unbroken cells and shear DNA.

Centrifugation etc. as in 3.

Solubilise the purified inclusion bodies into 6M guanidine HCl (Gnd-HCl) or 8M urea with appropriate buffer and 5-100mM DTT.

Some inclusion bodies are very difficult to solubilise and you might want to leave them to dissolve over-night.

Run a gel to check for the success of the inclusion body purification.

Refolding additives

Protein refolding from denatured proteins is influenced by several factors, including solubility of protein, removal of denaturant, and assistance of refolding additives. The addition of chemical additives has been frequently used to prevent protein aggregation. Chemical additives can have effects in the following aspects: stabilizing the natural structure of correct folded proteins, changing the stability of misfolded proteins, increasing the solubility of folding intermediates and un-folded molecules. Commonly used chemical additives are shown below:

Denaturants: urea and GdnHCl
Amino acids: Gly, Ala, Pro, and Arginine
Polymers: PEG and cyclodextrin
Polyols: glycerol
Alcohol: short-chain alcohols
Salts: NaCl, KCl, MgCl2 and (NH4)2SO4
Sugars: glucose, sucrose and trehalose
Detergents: Triton X-100, CHAPS, SDS and CTAB Detergents with cycloamylose or cyclodextran
Artificial chaperones: thioredoxin, disulfide bond isomerase
Reducing/oxidizing reagents: DTT, cysteine/cystine and glutathione
Non-detergent zwitterionic agents: Non-detergent sulfobetaines (NDSB)

Though different techniques have developed for the generation of correctly folded protein from solubilized inclusion bodies, there is no universal refolding recipe for any protein. Proteins folding in solution are affected by a number of physiochemical parameters including ionic strength, pH, temperature, oxidation state and protein concentration as well as the presence of hydrophobic, polar, chaotropic agents and other proteins. For any given protein, the best refolding conditions still have to be determined empirically. BiologicsCorp has rich experiences to solve problems of protein refolding in E.coli. After evaluation of target proteins' biochemical and the structural properties, BiologicsCorp can offer flexible mature solutions to solve problems of protein refolding.

On-column Refolding with beta-cyclodextrin

Inclusion body solubilization wascarried out in 8-M urea or 6-M guanidinium hydrochloride.Solubilized inclusion bodies were bound to Ni-NTA resin (Qi-agen, Valencia, California, USA) and pre-equilibrated in dena-turing buffer — by batch-absorption performed overnight atroom temperature. The amount of resin used is 5 mg of protein/1 ml of resin. On-column renaturation and purificationwereperformed the next morning by several changes of buffers.First, the column was washed using the denaturing buffer con-taining 20 mM imidazole to remove nonspecifically-boundcontaminants. beta-mercaptoethanol at a final concentration of10 mM was added to this buffer and all following buffers if therefolding protein contained cysteines.

In the next step, the column was washed with 10-column volumes (CV) of buffer A containing 0.1% Triton X-100 (buffer A (20 mM Tris-HCl, pH 8.0, 0.1 M NaCl). This was followed by a wash with 10 CV of Buffer A containing 5 mM beta-cyclodextrin (Sigma, St.Louis, Missouri, USA) to remove detergent from the pro-tein–detergent complex and to allow the protein to refold. Be-fore elution, an additional wash with 20 mM Tris-HCl, pH 8.0