Strategies for Native Protein and Recombinant Protein Purification Soluble Extracellular Proteins

SOLUBLE EXTRACELLULAR PROTEINS

The source of soluble extracellular proteins is the extracellular medium, whether it could be an animal source such as blood or spinal fluid, or a culture medium in which bacterial, fungal, animal, or plant cultures have been grown. Generally these do not contain a large number of different proteins (blood is an exception), and the desired protein may be a major component, especially if produced as the result of recombinant expression. Nonetheless, the protein in the starting material may be quite dilute, and a large volume may therefore need to be processed. The starting fluid may also contain many compounds other than proteins, whose behavior must be taken into account. The first stage should aim mainly to reduce the volume and get rid of as much nonprotein material as possible; some protein-protein separation is also useful, but not essential. No general rules can be given, but a batch adsorption method using an inexpensive material such as hydroxyapatite, ion-exchange resin, immobilized metal affinity chromatography (IMAC) medium, or affinity adsorbent is best, if feasible. Following the first step, the sample should be in a form that is amenable to standard purification processes such as precipitation and column chromatography.

INTRACELLULAR (CYTOPLASMIC) PROTEINS

To obtain soluble intracellular proteins (which are mainly enzymes), cells must be broken open or lysed to release their soluble contents. The ease with which cell disruption can be accomplished varies considerably; animal cells are readily broken, as are many bacteria, but plants and fungi have tough cell walls. The macromolecular soluble contents of cells are mainly proteins, with nucleic acids as a minor but significant component. Bacterial extracts may be viscous unless DNase is added to break down the long DNA molecules. Although chromatographic procedures can be applied to crude extracts, valuable highperformance materials should not be employed in the first step, as there are always compounds, including unstable proteins, that may bind to them and be difficult to remove.

MEMBRANE-ASSOCIATED PROTEINS

There are two approaches to isolating a membrane-associated protein. In one method, the relevant membrane fraction can first be prepared and then used to isolate the protein. Alternatively, whole tissue can be subjected to an extraction that solubilizes the membranes and releases the cytoplasmic contents as well. The former is much better in that purification is accomplished by isolating the membranes: the specific activity of the solubilized membrane fraction will be much higher than in the second method. However, the process of purifying the membrane fraction may lead to substantial losses, and it may be difficult to scale up. If total recovery of the protein is more important than purity, a whole-tissue extract is likely to be more appropriate. Although this means that a greater degree of purification is needed, the fact that membrane proteins have, by definition, properties somewhat different from those of cytoplasmic proteins permits some very effective purification steps (e.g., hydrophobic chromatography or fractional solubility separation). Peripheral membrane proteins are only loosely attached and may be released by gentle conditions such as high pH, EDTA, or low (nonionic) detergent concentrations. Once in solution, some peripheral proteins no longer require the presence of detergent to maintain their solubility. Integral membrane proteins are much more difficult-they require high concentrations of detergent for solubilization (i.e., complete solubilization of the membrane is needed to release them) and generally are neither soluble nor stable in the absence of detergent. It is sometimes necessary to maintain natural phospholipids in association with the proteins in order to maintain activity. Even though the final objective may not require activity (e.g., amino acid sequencing), there is a need for some sort of assay during the purification process to determine where the protein is. If a particular band on a gel is known to be the desired protein, then no other assay is needed and loss of bioactivity can be allowed. Purification processes may be affected by the presence of detergents. The problem of association with detergent micelles makes purifying integral membrane proteins difficult; the close association of the different proteins originating from membranes often results in very poor separation in conventional fractionation procedures.

INSOLUBLE PROTEINS

Natural proteins that are insoluble in normal solvents are generally structural proteins, which are sometimes cross-linked by posttranslational modification. The first stage of purification is obvious-it involves extracting and washing away all proteins that are soluble, leaving the residue containing the desired material. Further purification in a native state, however, may be impossible; extracting away other proteins using more vigorous solvents or attempting to solubilize the target protein may destroy the natural structure. Cross-linked proteins such as elastin or aged collagen cannot be dissolved without breaking the cross-links, and the individual proteins may even be crosslinked together.

INSOLUBLE RECOMBINANT PROTEINS (INCLUSION BODIES)

A major new class of insoluble proteins are recombinant proteins expressed (usually in Escherichia coli) as inclusion bodies. These are dense aggregates found inside cells that consist mainly of a desired recombinant product, but in a nonnative state. Inclusion bodies may form for a variety of reasons, such as insolubility of the product at the concentrations being produced, inability to fold correctly in the bacterial environment, or inability to form correct, or any, disulfide bonds in the reducing intracellular environment. Their purification is simple, since the inclusion bodies can be separated by differential centrifugation from other cellular constituents, giving almost pure product; the problem is that the protein is not in a native state, and is insoluble. Some methods for obtaining an active product from inclusion bodies are described in following.

SOLUBLE RECOMBINANT PROTEINS

Recombinant proteins that are not expressed in inclusion bodies either will be soluble inside the cell or, if using an excretion vector, will be extracellular (or, if E. coli is the host, possibly periplasmic). They can be purified by conventional means. In some systems, expression is so good that the desired product is the major protein present and its purification is relatively simple. In systems where the expression level is low, the purification process can be tedious, though easier, it is hoped, than isolation from the natural source. It should be remembered that a procedure developed for isolating a protein from natural sources may not work successfully with the recombinant product, because the nature of the other proteins present influences many fractionation procedures. Because of the difficulties often experienced in purifying recombinant proteins, a variety of vector systems have been developed in which the expressed prod-uct is a fusion protein containing an N-terminal polypeptide that simplifies purification. Such "tags" can be subsequently removed using a specific protease. A further advantage is that the expression level is dictated mainly by the transcription and translation signals for the fusion portion of the protein, which are optimized. Tags used include proteins and polypeptides for which there is a specific anti- body, binding proteins that will interact with columns containing a specific ligand, polyhistidine tags with affinity to immobilized metal columns, sequences that result in biotinylation by the host and enable purification on an avidin column, and sequences that confer insolubility under specified conditions. Unstable proteins may be modified by the molecular biological technique of site-directed mutagenesis to remove the site of instability- for instance, an oxidizable cysteine. Such techniques are appropriate for commercial production of proteins, but may of course alter natural functioning parameters. Increased thermostability can be one modification, although it is not easy to predict mutations that will improve that parameter. Thermostable proteins originating from thermophilic bacteria do not need structural modification and, if expressed in large amounts, can be purified satisfactorily in one step by simply heat-treating the extract at 70¡ãC for 30 min, which denatures virtually all the host proteins (e.g., see Oka et al., 1989). The host bacteria used for production of recombinant proteins are usually Escherichia coli, or Bacillus subtilis; they may express proteins at 1% to over 50% of the cellular protein, depending on such variables as the source, promoter structure, and vector type. Generally the proteins are expressed intracellularly, but leader sequences for excretion may be included. In the latter case, the protein is generally excreted into the periplasmic space, which limits the amount that can be produced. Excretion from gram-positive species such as B. subtilis sends the product into the culture medium, with little feedback limitation on total expression level.

---

©2013 BiologicsCorp, All right reserved.

Contact Us