The ability to clone and over-express genes to obtain proteins from any source organ ism has so many advantages that the starting material will therefore most likely be from one kind of cell culture, as discussed above. However, post-translational modifications, poor folding or poor induction characteristics can still make it necessary in some instances to purify native proteins of interest from an abundant source. Protein purification procedures necessarily start with the disruption of cells or tissue to release the protein content of the cells into an appropriate buffer, unless the protein is secreted. This initial extract is therefore the starting point for protein purification, but thought must first be given to the composition of the buffer used to extract the proteins.

Extraction Buffer

 Normally, extraction buffers are at an ionic strength of 0.1–0.2 M of a monovalent salt and a pH between 7 and 8, which is considered to be compatible with the conditions found inside the cell. TRIS or phosphate buffers are most commonly used. Additionally, a range of other reagents may be added to the buffer for specific purposes. These include:

• An antioxidant : Within the cell, proteins are in a fairly reducing environment, but when released into the buffer they are exposed to a more oxidising environment. Since most proteins contain a number of free thiol groups (from the amino acid cysteine) these can undergo oxidation to give inter- and intramolecular disulfide bridges. To prevent this, reducing agents such as dithiothreitol, β-mercaptoethanol, cysteine or reduced glutathione are often included in the buffer.

• Enzyme inhibitors: Once the cell is disrupted, the organisational integrity of the cell is lost, and proteolytic enzymes that were carefully packaged and controlled within the intact cells are released, for example from lysosomes. Such enzymes may degrade proteins in the extract, including the protein of interest. To slow down unwanted proteolysis, all extraction and purification steps are carried out at 4 °C, and, in addition, a range of protease inhibitors is included in the buffer. Each inhibitor is specific for a particular type of protease, for example serine proteases, thiol proteases, aspartic proteases and metalloproteases. Common examples of inhibitors include: di-isopropylphosphofluoridate (DFP), phenylmethyl-sulfonylfluoride (PMSF) and tosylphenylalanyl-chloromethylketone (TPCK) (all serine protease inhibitors); iodoacetate and cystatin (thiol protease inhibitors); pepstatin (aspartic protease inhibitor); EDTA and 1,10-phenanthroline (metalloprotease inhibitors); and amastatin and bestatin (exopeptidase inhibitors).

 • Enzyme substrate and cofactors: Low levels of substrate are often included in extraction buffers when an enzyme is purified, since binding of substrate to the enzyme active site can stabilise the enzyme during purification processes. Where relevant, cofactors that otherwise might be lost during purification are also included to maintain enzyme activity so that it can be detected when column fractions are screened.

• Phosphatase inhibitors : Sodium orthovanadate (Na₃VO₄) is a general inhibitor of phosphotyrosyl phosphatases, commonly used in buffers for protein kinase assays. Being a competitive inhibitor, it can be conveniently removed by dilution, inactivation with EDTA or dialysis. Na₃VO₄ is often combined with sodium fluoride, which inhibits phosphoseryl and phosphothreonyl phosphatases. β-glycerol-phosphate is a general serine/threonine phosphatase inhibitor that acts as a pseudo-substrate by pro viding a ready source of organic phosphate.

 • EDTA: This is used to chelate divalent metal cations; each molecule of EDTA will bind one cation. The binding is very efficient due to the coordination provided by four oxygen and two nitrogen atoms. EDTA is added to remove divalent metal ions (M²⁺) that can react with thiol groups in proteins giving thiolates as per:

• The sequestration of free magnesium ions by EDTA decreases the activity of any enzyme that uses ATP as a cofactor. Cation chelation also reduces the activity of metal loproteases. However, EDTA would obviously interfere with immobilized metal affinity chromatography (IMAC) used to purify His-tagged proteins. Additionally, EDTA inter feres with nucleotide binding.

• Polyvinylpyrrolidone (PVP): This is often added to extraction buffers for plant tissue. Plant tissue contains considerable amounts of phenolic compounds (both monomeric, such as p -hydroxybenzoic acid, and polymeric, such as tannins) that can bind to enzymes and other proteins by non-covalent forces, including hydrophobic, ionic and hydrogen bonds, causing protein precipitation. These phenolic compounds are also easily oxidised, predominantly by endogenous phenol oxidases, to form quinones, which are highly reactive and can combine with reactive groups in proteins causing cross-linking, and further aggregation and precipitation. Insoluble PVP (which mimics the polypeptide backbone) is therefore added to adsorb the phenolic compounds, which can then be removed by centrifugation. Thiol compounds (reducing agents) are also added to minimise the activity of phenol oxidases, and thus prevent the formation of quinones.

• Sodium azide : For buffers that are going to be stored for long periods of time, antibacterial and/or antifungal agents are sometimes added at low concentrations. Sodium azide is frequently used as a bacteriostatic agent.

Membrane Proteins

 Membrane-bound proteins require special conditions for extraction as they are not released by simple cell-disruption procedures alone. Two classes of membrane proteins are identified. Extrinsic (or peripheral) membrane proteins are bound only to the surface of the cell, normally via electrostatic and hydrogen bonds. These proteins are predominantly hydrophilic in nature and are relatively easily extracted, either by raising the ionic concentration of the extraction buffer (e.g. to 1 M NaCl) or by either acidic or basic conditions (pH = 3–5 or pH = 9–12). Once extracted, the peripheral membrane proteins can be purified by conventional chromatographic procedures. Intrinsic membrane proteins are those that are embedded in the membrane. These invariably have significant regions of hydrophobic amino acids (those regions of the protein that are embedded in the membrane, and associated with lipids) and have low solubility in aqueous buffer systems. Hence, once extracted into an aqueous polar environment, appropriate conditions must be used to retain their solubility. Intrinsic proteins are usually extracted with buffer containing detergents. The choice of detergent is mainly one of trial and error, but can include ionic detergents such as sodium dodecyl sulfate (SDS), sodium deoxycholate, cetyl trimethylammonium bromide (CTAB) and CHAPS, and non-ionic detergents such as Triton X-100 and Nonidet P-40.

Once extracted, intrinsic membrane proteins can be purified using conventional chromatographic techniques such as size-exclusion, ion-exchange or affinity chromatography (using lectins). Importantly, in each case, it is necessary to include detergent in all buffers to maintain protein solubility. The level of detergent used is normally 10- to 100-fold less than that used to extract the protein, in order to minimise any interference of the detergent with the chromatographic process.

Sonication

This method is ideal for a suspension of cultured cells or microbial cells. A sonicator probe is lowered into the suspension of cells and high frequency sound waves ( <20 kHz, i.e. ultrasound) generated for 30–60 s. These sound waves cause disruption of cells by shear force and cavitation. Cavitation refers to areas where there is alternate compression and rarefaction, which rapidly interchange. The gas bubbles in the buffer are initially under pressure but, as they decompress, shock waves are released and disrupt the cells. This method is suitable for relatively small volumes (50–100 cm³). Since considerable heat is generated by this method, samples must be kept on ice during treatment, typically in a so-called rosette cell. The efficiency of cell lysis by sonication is thought to be around 50–60%. The possible danger of damaging the target protein is a disadvantage of this method.

Repeated Freeze–Thaw

A more gentle and readily available method for lysis of bacterial cells is to subject the aqueous cell suspension to repeated freezing and thawing conditions. Resuspended cells are frozen (either at −20 °C, −80 °C or in liquid nitrogen) and then thawed in warm water. This procedure is repeated three times. The shear forces due to ice formation cause disruption of the enclosed cells. Frequently, a short sonication of the resulting homogenate (lysate) is carried out to break down the bacterial genomic DNA, which can make the suspension stringy and viscous.

Blenders

These are commercially available, although a typical domestic kitchen blender will suffice. This method is ideal for disrupting mammalian or plant tissue by shear force. Tissue is cut into small pieces and blended, in the presence of buffer, for about 1 min to disrupt the tissue. This method is inappropriate for bacteria and yeast, but a blender can be used for these microorganisms if small glass beads are introduced to produce a bead mill. Cells are trapped between colliding beads and physically disrupted by shear forces.

Grinding With Abrasives

 Grinding with a pestle in a mortar, in the presence of sand or alumina and a small amount of buffer, is a useful method for disrupting bacterial or plant cells; cell walls are physically ripped off by the abrasive. However, the method is appropriate for handling only relatively small samples. The Dyno ® -mill is a large-scale mechanical version of this approach; it comprises a chamber containing glass beads and a number of rotating impeller discs. Cells are ruptured when caught between colliding beads. A 600 cm 3 laboratory scale model can process 5 kg of bacteria per hour.

Presses

The use of homogenisers such as a French press, or the Manton–Gaulin press, which is a larger-scale version, is an excellent means for disrupting microbial cells. A cell suspension (~50 cm³ ) is forced by a piston-type pump under high pressure (10 000 psi ≈ 1450 kPa) through a small orifice. Breakage occurs due to shear forces as the cells are forced through the small orifice, and also by the rapid drop in pressure as the cells emerge from the orifice, which allows the previously compressed cells to expand rapidly and effectively burst. Multiple passes are usually needed to lyse all the cells, but under carefully controlled conditions it is possible to selectively release proteins from the periplasmic space. The Hughes press is a variation on this method; the cells are forced through the orifice as a frozen paste, often mixed with an abrasive. Both the ice crystals and the abrasive aid in disrupting the cell walls.

Enzymatic Methods

The enzyme lysozyme, isolated from hen egg whites, cleaves peptidoglycan. The peptidoglycan cell wall can therefore be removed from Gram-positive bacteria (see Figure 1) by treatment with lysozyme, and if carried out in a suitable buffer, the cell mem brane will rupture, owing to the osmotic effect of the suspending buffer.

Fig1. The structure of the cell wall of Gram-negative and Gram-positive bacteria. LPS, lipopolysaccharide.

Gram-negative bacteria can similarly be disrupted by lysozyme, but treatment with EDTA (to remove divalent metal ions, thus destabilising the outer lipopolysaccharide layer) and the inclusion of a non-ionic detergent to solubilise the cell membrane are also needed. This effectively permeabilises the outer membrane, allowing access of the lysozyme to the peptidoglycan layer. If carried out in an isotonic medium so that the cell membrane is not ruptured, it is possible to selectively release proteins from the periplasmic space.

Yeast can be similarly disrupted using enzymes to degrade the cell wall, followed by either osmotic shock or mild physical force to disrupt the cell membrane. Enzyme digestion alone allows the selective release of proteins from the periplasmic space. The two most commonly used enzyme preparations for yeast are zymolyase or lyticase, both of which have β-1,3-glucanase activity, together with a proteolytic activity specifi c for the yeast cell wall. Chitinase is commonly used to disrupt filamentous fungi. Enzymatic methods tend to be used for laboratory-scale work, since for large-scale work their use is limited by cost.

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