After producing the proteins of interest in suitable expression systems or after gaining sufficient amounts from natural sources, the protein of interest must then be separated from any contaminating substances such as sugars, lipids, and/or

Fill tube

Exhaust vent filter

Inlet air filter

Wave Bioreactor Version

Inlet air filter

Figure 3 Wave Bioreactor System (Wave Biotech AG): the figure shows a cell bag filled with medium and cells on a wave bioreactor rocking platform.

Sterile cellbag - Sampling device Temperature control

Control of aeration, shaking

Figure 3 Wave Bioreactor System (Wave Biotech AG): the figure shows a cell bag filled with medium and cells on a wave bioreactor rocking platform.

other proteins. There is no general procedure for protein preparation and a wide range of literature is available on that subject (see Section 3.19.1). In most cases, it consists of the following steps:

• centrifugation

• a least one (column) chromatography step for purification

• concentration

• buffer exchange

Mostly, the purification procedure consists of three steps:

1. capture phase for quick isolation and stabilization of the protein

2. intermediate purification procedure for removal of bulk impurities

3. polishing to achieve over 90% purity levels when separating the protein of interest from trace contaminants and/or closely related substances

Over the last decade, developments in recombination techniques have revolutionized protein production in many ways. Engineered proteins have been designed with improved solubility and protein properties and simplified purification with the help of affinity tags. It has to be mentioned that these protein mutations may interfere with folding, function, or crystallizability of the protein of interest.

In bacterial expression systems, refolding of inclusion bodies protein sometimes requires additional time-consuming steps, together with a reliable and robust test of molecular activity of the protein of interest. Cell Lysis

All proteins that are produced intracellularly require cell disruption for release. Depending on the expression system used, different methods of breaking up the expression hosts are necessary. When proteins are secreted into the medium by the usage of secretion signals, no cell lysis is necessary. Fractionated precipitation or ultrafiltration can be performed to concentrate the protein of interest out of the large volume of cell medium. Proteins expressed in the periplasm can be isolated by mild osmotic shock.

Cells from mammalian tissue origin are destroyed by mechanical procedures such as homogenizers or potters. Mammalian and insect cells from cell cultures have to undergo freeze/thaw cycles or mild detergent treatment.

Bacterial cell membrane protection is more stable: treatment with lysozyme destabilizes the cell by hydrolysis of the 1,4 glycosidic bonds in the peptidoglycan cell wall. Subsequent detergent treatment releases most of the soluble content of the cells. Sonication, French Press, Constant Cell Disruption System (Constant System Ltd., Daventry, Northants, NN114SD, UK), or bead mill treatment are also very effective cell disruption methods.

Yeast cells have to go through harsh treatment (French Press or cell mill with glass beads) to undergo lysis. Independently of the chosen lysis procedure, the addition of protease inhibitors, reducing agents, and glycerol to the lysis buffer helps to stabilize the protein and protects it against degradation.

After lysis, cell membrane fragments and cells with other insoluble or large particles have to be separated from the soluble protein by centrifugation (40 000 g for 30-60 min) and filtering it through 0.2 mm pore size filter. Refolding

Proteins produced in a microbial host at high expression levels are often produced in an unfolded or improperly folded form and then accumulate in intracellular vesicles called inclusion bodies.52 Inclusion bodies consist of more than 90% of the protein of interest. After gaining this nearly pure protein fraction, refolding has to be performed to obtain active protein suitable for structural studies.

The process of refolding consists of three steps: (1) total denaturation of the protein; (2) slow renaturation (duration: hours to weeks.); and (3) the gain of the correctly folded protein (yields: 5-40% of the total inclusion bodies protein) by column chromatography (for a review on refolding, see Rudolph etal. ). Inclusion bodies purification

To separate inclusion bodies protein from soluble protein and cell components, extensive washing steps (resuspension of lysis pellet in different solutions followed by centrifugation to pellet the inclusion bodies protein) are necessary. Suitable buffers for washing contain detergent (solubilizes membrane fragments), high salt concentrations (salt solubilizes proteins), and up to 1 M urea. Solubilization of inclusion bodies protein

After washing, the pelleted inclusion bodies protein has to be solubilized and totally denatured. The pellet is homogenized in a buffer containing a chaotropic agent such as 6M guanidinium hydrochloride or 8M urea and dithiothreitol (DTT) or b-mercaptoethanol (b-ME), which will reduce disulfide bonds (for proteins containing more than one cysteine residue) and is then incubated for l h at 37 °C. After that procedure, insoluble parts are to be removed by centrifugation. Refolding conditions and screens

Fast dilution of the denatured protein in, or slow dialysis against, an appropriate refolding buffer reduces the concentration of the chaotropic denaturant. This procedure takes several hours to several weeks and produces a mixture of different folding conditions of the protein of interest: A larger amount will fold incorrectly, precipitate during the refolding procedure, and can then be easily removed by means of centrifugation or expanded bed chromatography. Another amount will exhibit incorrect folding but will stay in solution and will then require separation from the correctly folded protein by means of chromatography.

Different proposals for refolding screens are to be found in the literature; the main components of refolding buffers are arginine or sucrose (which act as stabilizing agents); sodium chloride or potassium chloride as ionic molecules; detergents for solubilizing hydrophobic patterns of the protein; low concentrations of urea or guaninidium hydrochloride as chaotropes; reducing/oxidizing agent mixture for correct formation of disulfide bonds (oxidized and reduced glutathion); pH and temperature variations.54-57

After refolding, the insoluble, incorrectly folded proteins are to be removed by centrifugation. Any remaining impurities and soluble but incorrectly folded protein molecules are to be removed by column chromatography. Capture Step with Affinity Tags

In the last decade, a broad variety of fusion proteins or peptides for simplified protein purification and detection have been described. The fusion partner is encoded on the plasmid or viral DNA and either C- or N-terminally linked to the protein of interest. For larger fusion tags the introduction of an appropriate protease cleavage site between fusion protein and the protein of interest is considered to remove the tag prior to crystallization. Careful removal of the protease before starting crystallization experiments is very important so as to avoid cleavage of the protein of interest during the crystallization procedure.

Plasmids containing DNA coding for proteins like glutathione-S-transferase,58'59 maltose-binding protein, NusA, calmodulin-binding peptide, intein, thioredoxin, cellulose-binding protein, and Fc fragments of antibodiesl7 are commercially available, together with the appropriate protein purification tools. Many of these large fusion partners increase the solubility of the expressed fusion protein (see, for example, Kapust and Waugh60). Sometimes fusion partners simulate solubility of the expressed protein. After removal of the tag, the protein of interest precipitates irreversibly.

Small affinity tags such as Poly-His-tag and Strep-tag6^62 are widely used as they do not change protein properties and do not need to be cleaved off. When using Poly-His-tags for protein purification, it has to be taken into account that E. coli strains produce protein SlyD (20kDa) containing a potential metal-binding site domain. This protein binds tightly to metal chelate chromatography and is often detected as a contamination band.63

Affinity tag chromatography can be performed in batch mode, by centrifugal or syringe filter devices, or on chromatographic equipment with prepacked columns according to the manufacturer's instructions.58,62,64

In some cases, removal of the elution agent is necessary to maintain protein stability: Fc-fragment fusion proteins are eluted in extreme acid conditions: pH has to be adjusted to neutral values immediately after elution. Using imidazole in high concentrations in the elution of Poly-His-tagged proteins destabilizes proteins and it has to be removed by dialysis. Fractionated Precipitation

Fractionated precipitation with ammonium sulfate or alcohol has been a widely used method for tissue or plasma protein purification.65 Solubility of proteins depends on different parameters, such as temperature, pH, salt, and protein concentration, and is lowest at pH values of around the isoelectric point of the protein. Therefore, different proteins precipitate at different conditions. This can be used as a fast and quantitative protein purification step, especially for proteins secreted to the medium.

Precipitation of proteins with 1-4M ammonium sulfate or 10-40% (w/v) polyethyleneglycol (PEG) under pH and temperature control is a convenient method for fast increase of protein concentration. The precipitate is dissolved in a small amount of solubilizing buffer and dialyzed against the same buffer to regain solubility and perform the next purification step. Low-Pressure Column Chromatography

Column chromatography covers protein separation steps performed on chromatography columns filled with a variety of column matrices (mostly derivatives of agarose) using different protein characteristics for separation. The columns require pumps to be run and an ultraviolet detector for protein determination. Complete equipment for column chromatography from either GE Healthcare or BioRad provides additional pH and conductivity monitors and fraction collectors.

Protein characteristics used for separation are size, charge, affinity toward substrates or inhibitors, hydrophobicity, lectin, or antibody binding. The most prominent column chromatography methods are described below.

A variety of simplified affinity and ion exchanger-binding tools are available on the market. These are tools that do not require chromatography equipment but are used in a batch mode or as centrifugal devices. The separation results and protein-binding capacities are not comparable to chromatography methods, but give quick, low-cost results for pre-studies.

Before starting column chromatography, clearance of the protein sample and the use of centrifugation and filtration to remove all dust and cellular particles are indispensable. Affinity chromatography

The most efficient purification method is column chromatography with an affinity medium, although it is not applicable in all cases. Similar to the affinity-tag purification capture step mentioned above (Section, affinity chromatography binds proteins with respect to their specific and natural properties.

The user must have comprehensive knowledge of the protein of interest and how it functions or reacts enzymatically, any possible cofactors or substrates, and/or about its sequence and modification pattern.

A very specific, but expensive, method of affinity chromatography is purification by specific antibodies coupled to column matrices. The binding to these matrices is performed at neutral pH values while elution of the bound protein of interest requires pH values of around 2-3, which is not suitable for every protein, but very effective when possible.

The same holds for capturing enzymes to specific inhibitors, cofactors, or substrate analogs coupled via activated groups to agarose: elution can be performed by the addition of the coupled substance in a free form. Commercially available examples such as adenosine triphosphate or NADPH agarose for capturing kinases, lectin matrices for the capture of glycoproteins, or heparin agarose for binding of blood coagulation factors are widely used.

Capturing the protein of interest by its binding to specific inhibitors, peptides, or substrate analogs chemically bound to activated agaroses is very efficient. These affinity chromatographies are very helpful, particularly for removing randomly folded protein from correctly folded ones, after the refolding procedure. Proteins and peptides can be coupled via amino groups to cyanogen bromide or 6-aminocaproic acid N-hydroxysuccinimide ester-activated-Sepharose, or via carboxyl groups to aminohexyl Sepharose. Many other matrices with a variety of functional groups for protein and substrate coupling are commercially available.4 Ion exchange chromatography

Ion exchange chromatography matrices capture proteins from a solution by ionic interaction. The overall isoelectric point (IP) can be calculated from the amount and Ki values of all charged amino acid residues in a protein. At buffer pH values above this IP, the protein is negatively charged (anionic); at pH values below that, the protein is positively charged (cationic).

For the binding of anionic proteins, anion exchanger matrices such as quaternary amines or diethylaminoethyl groups coupled via a linker to a cellulose matrix are available.

For the binding of cationic proteins, sulfopropyl or carboxymethyl groups can be used. The proteins are eluted by a sodium chloride gradient providing increasing ionic strength to break the interaction of protein with charged matrix.

The proteins bind at suitable pH values in the absence of ionic substances such as salts. Usually, a linear salt gradient from 0 to 1 M is used for stepwise elution of contaminating proteins and proteins of interest. Depending on the diversity and amount of contaminants, one-step ion exchange chromatography may lead to purity that is sufficient for structural studies. Microheterogeneities of the protein of interest (due to posttranslational modifications such as glycosylation or phosphorylation or to aggregation status) result in multiple-peak elutions. These different protein homologs require separation from each other prior to crystallization. Hydrophobic interaction chromatography

Protein mixtures can be separated by their different hydophobic/hydrophilic natures through hydrophobic interaction chromatography. The proteins are applied on the column matrix carrying hydrophobic ligands (phenyl-, ethyl, or alkyl residues) in a solution containing a high percentage of hydrophilic substances (e.g., >1.5M ammonium sulfate), to decrease the water solubility of the proteins to a minimum. Elution is performed by the stepwise decrease of the hydrophilic substance. Size exclusion chromatography (SEC)

For the separation of proteins with a high molecular mass from those with a low one, SEC is the method of choice. As SEC columns do not have high separation capacities, this method is not suitable as a capture, but only for the last, polishing step in the protein purification procedure. A high degree of caution is necessary in the choice of running buffer: its pH value has to differ by at least 1 pH unit from the IP of the protein. Additionally, the NaCl concentration needs to be above 150 mM to avoid unspecific interference to the column matrix. Unless it is necessary for protein solubilization, detergent should be avoided so as to prevent partial denaturation of the protein.

SEC columns are restricted in the volume of the sample to be applied, depending on the column diameter and length. For this reason, concentration of the protein sample prior to application on the SEC column is recommended. The smaller the sample volume and flow rate are, the better the separation results.

Before starting crystallization experiments, SEC is recommended to determine the aggregation status of the protein (Figure 4). Monomeric proteins or protein aggregations of defined size crystallize more easily than undefined aggregate mixtures. Buffer Exchange

Before starting the affinity process, hydrophobic or ion exchange chromatography, or crystallization experiments, the protein sample has to be converted to defined buffer conditions, depending on the nature of the protein in hand and the method to be performed. Different methods are available for changing the buffer but it is imperative not to dilute the protein sample too far. The most effective, mildest, but also most time-consuming method is dialysis. Dialysis

A variety of dialysis tools are on the market: regenerated cellulose tubings, Slide-A-Lyzer,67 tube-O-Dialyzer,68 and dialysis buttons,69 all of which work on the same principle: the protein sample is transferred to a container with a semipermeable membrane (usually cellulose) closure. By means of this membrane, small molecules such as buffer

0 50 100 150 200 250 300

Figure 4 Size exclusion chromatography on HiLoad 26/60 Superdex 75 pg on fast performance liquid chromatography (FPLC) (GE Healthcare Amersham Biosciences58). Peak 1, cytochrome c, 1 mgmL-1, Sigma C-3006, molecular weight 12.4 kDa. Peak 2, carbonic anhydrase, 1 mg mL-1, Sigma C-7025, molecular weight 29kDa. Peak 3, bovine serum albumin, 1 mgmL-1 Sigma P0914, molecular weight 67 kDa. Peak 4, bovine serum albumin dimer, 1 mgmL-1 Sigma P0914, molecular weight 134 kDa. Peak 5, bovine serum albumin tetramer, 1 mg mL-1 Sigma P0914, molecular weight 268 kDa.

0 50 100 150 200 250 300

Figure 4 Size exclusion chromatography on HiLoad 26/60 Superdex 75 pg on fast performance liquid chromatography (FPLC) (GE Healthcare Amersham Biosciences58). Peak 1, cytochrome c, 1 mgmL-1, Sigma C-3006, molecular weight 12.4 kDa. Peak 2, carbonic anhydrase, 1 mg mL-1, Sigma C-7025, molecular weight 29kDa. Peak 3, bovine serum albumin, 1 mgmL-1 Sigma P0914, molecular weight 67 kDa. Peak 4, bovine serum albumin dimer, 1 mgmL-1 Sigma P0914, molecular weight 134 kDa. Peak 5, bovine serum albumin tetramer, 1 mg mL-1 Sigma P0914, molecular weight 268 kDa.

ingredients, salts and detergents permeate, protein, or other molecules larger than the molecular weight cut-off of the dialysis membrane will remain in the container. Defined buffer conditions are obtained after approximately 6 h time and two exchanges of the dialysis buffer (volume 100 x sample volume). Size exclusion chromatography

SEC for buffer exchange can either be performed as described above (in Section at conventional column chromatography workstations or with low-cost and low-tech variations of the same method: PD10/NAP-5,58 zip-tips for MS,70 bio -spin chromatography columns.71 Caution has to be taken as described above (in Section to choose the right buffer to avoid unspecific interactions with the column materials. The protein sample will then, afterwards, have been diluted. Ultrafiltration

The fastest method of exchanging buffers and simultaneously concentrating protein samples is ultrafiltration. Again, many tools with different molecular weight cut-offs (1-100 kDa) are offered on the market: Centrifugal devices of different sizes; N2 pressure-driven stirring cells or pump driven crossflow filtration systems.66'70'72 The buffer of the protein sample is pressed through a membrane permeable for low-molecular-weight components: the proteins remain in the sample chamber. After several refills, buffer exchange is more than 99% complete. Concentration

Protein crystallization and NMR spectroscopy experiments need high protein concentrations (usually around 10mgmL_ 1). For that reason, concentration of the purified protein prior to structure analysis methods is necessary. Concentration steps have to be performed with caution, as many proteins tend to precipitate at very high concentrations (> 20mgmL_ 1), in low-ionic-strength solutions. Concentration with polyethyleneglycol

PEG has a very high water-binding capacity. Embedding dialysis tubing filled with protein solution, in solid PEG (molecular weight of the PEG larger than the cut-off of the dialysis membrane) draws the buffer out of the sample and concentrates the protein in a very slow and nonreactive manner. Ultrafiltration

Ultrafiltration procedures, as described above (in Section, with centrifugal, nitrogen, or pump-driven devices, are the most common methods used to increase protein concentration.

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook

Post a comment