Purification Protocols

In the following, we will describe our current protocols for isolating staphylococcal alpha-toxin and streptolysin-O from bacterial culture supernatants. The protocol for isolating recombinant streptolysin-O from call has been published (Weller et al., 1996) and will not be 

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In particular, the availability of such bacterial biocatalysts in the form of recombinant expression systems [136] in combination with simplified purification protocols opened up this methodology for large-scale applications [204]. 

Figure 3 Purification protocol of imidazolium ionic liquid.

The quantity, quality and purity of the template DNA are important factors in successful PGR amplification. The PGR is an extremely sensitive method capable of detecting trace amounts of DNA in a crop or food sample, so PGR amplification is possible even if a very small quantity of DNA is isolated from the sample. DNA quality can be compromised in highly processed foods such as pastries, breakfast cereals, ready-to-eat meals or food additives owing to the DNA-degrading action of some manufacturing processes. DNA purity is a concern when substances that inhibit the PGR are present in the sample. For example, cocoa-containing foodstuffs contain high levels of plant secondary metabolites, which can lead to irreversible inhibition of the PGR. It is important that these substances are removed prior to PGR amplification. Extraction and purification protocols must be optimized for each type of sample. 

Poly(HA) depolymerases - as far as it has been tested - do not bind to anion exchangers such as DEAE (at neutral pH) but have a pronounced affinity to hydrophobic materials. Therefore, many purification protocols include hydrophobic interaction chromatography. 

Figure 1.2 Schematic outlining the purification protocol. The principal steps of purifying FLAG-tagged eIF2B proteins from yeast whole cell extracts are outlined in the diagram for details, refer to the main text.

It is useful to subdivide the downstream processing of recombinant proteins into a few key stages, often referred to as initial processing of the source material and extraction (if necessary), capture, intermediate purification and polishing. These can then be further split into unit operations. In each of these stages, predefined goals have to be achieved, so a well-defined purification protocol will sequentially utilize as... 

The physicochemical and other properties of any newly identified drug must be extensively characterized prior to its entry into clinical trials. As the vast bulk of biopharmaceuticals are proteins, a summary overview of the approach taken to initial characterization of these biomolecules is presented. A prerequisite to such characterization is initial purification of the protein. Purification to homogeneity usually requires a combination of three or more high-resolution chromatographic steps (Chapter 6). The purification protocol is designed carefully, as it usually forms the basis of subsequent pilot- and process-scale purification systems. The purified product is then subjected to a battery of tests that aim to characterize it fully. Moreover, once these characteristics have been defined, they form the basis of many of the QC identity tests routinely performed on the product during its subsequent commercial manufacture. As these identity tests are discussed in detail in Chapter 7, only an abbreviated overview is presented here, in the form of Figure 4.5. 

Table 6.2 Chromatographic techniques most commonly used in protein purification protocols. The basis of separation is listed in each case...

The advantage of such co-purification protocols is that the fully processed protein serving as the bait can allow interactions in a native environment and cellular location to allow isolation of multicomponent complexes. One limitation with this approach is the necessity for an antibody with specific immunoreactivity and immunoprecipitative capability for the bait protein. This drawback can be addressed by expression of the protein with an epitope tag. Excellent antibodies to a variety of epitope tags are available and can be utilized for immunoaffinity purification. Tags such as 6-histidine and GST allow purification using affinity characteristics to nickel and GSH beads, respectively. 

When making pharmaceuticals, one critical issue is to control and minimize metal impurities in the product, often to less than 10 ppm. Each product requires a different work-up and purification protocol, and it is difficult to describe a general solution. On some occasions washing removes the catalyst, but at other times the product is crystallized and the catalyst remains in the mother liquors occasionally, the product is volatile and can be distilled. Sometimes the catalyst is carried forward to the next stage and is removed at this point. In our experience, residual metal has not been problematic, but if it is then either immobilized or water-soluble catalysts, as described in this chapter, can be employed. 

The purification of solubilized proteins involves standardized protein purification protocols. Because the literature already contains a copious supply of available reviews and monographs regarding protein purification (35-43), the topic will not be discussed here. The reader is directed to ref. (38) for techniques specifically applicable to membrane proteins. 

Doonan S. Protein Purification Protocols, Humana Press, Totowa, NJ, 1996. 

The first problem encountered once the peptide has been successfully synthesized is that standard purification protocols fail. Although very hydrophobic peptides are soluble in acids such as TFA, these harsh conditions are not suitable for purification, because they can reduce column life times and denature native protein structures. Hence residual acid has to be removed, and many peptides can then be redissolved in mixtures of water and tert-butanol. Peptides with a strong tendency to aggregate may be dissolved either in trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), mixtures of 1-propanol and 1-butanol, 20% acetic acid or 70-90% formic acid. 

Because of the speed and high resolution of CZE separations as well as the small sample volumes required to yield information about complex protein samples, CE is increasingly being used to assess protein purity in multistep purification protocols in laboratory, pilot plant, and process scales. Similarly, it is being considered as a candidate for monitoring fermentation. 

Figure 10.2 Optimization of a purification protocol by LC-MS analysis. (1) LC-MS analysis of C-terminal fragment from reference molecule purified by cation-exchange chromatography (CEX) and hydroxyapatite chromatography (HA). (2) Incubation at 37°C under acidic conditions shows degradation of the purified molecule if CEX precedes HA. (3) The molecule is stabilized when the sequence of the two purification steps is swapped. 

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

For design of a simple manufacturing process, the thermostability of the NP enzymes is a very useful feature. Although heat treatment can be used as part of a purification protocol to isolate the enzymes from contaminating materials, the high temperature of operation itself excludes undesired enzyme-catalysed side reactions. For example, in the synthesis of 9-p-D-arabinofuranosyladenine from Ara-U and adenine, using a wet cell paste of Enterobacter aerogenes, adenine and Ara-U mainly underwent deamination at lower temperatures to form hypoxanthine and uracil respectively. At elevated temperature, deamination was completely eliminated and the rate of transarabinosylation increased. 

The initial procedures involved in expression of the Rhodnius nitrophorins in Escherichia coli were worked out for NPl by Drs. Donald E. Champagne and John F. Andersen at the University of Arizona during the spring and summer of 1996, and the purification protocols were... 

Structure of a HAT from the p300/CBP family has been long awaited, since these enzymes have no sequence similarity with enzymes of the GNAT and MYST families. The structure of the p300 HAT domain was solved very recently in complex with a Lys-GoA bi-substrate inhibitor, which required a highly intricate expression and purification protocol in order to produce a protein amenable to crystallization studies [24]. Analysis of the structure revealed however that, despite no sequence conservation, a similar structural core is observed as for the GNAT and MYST... 

In liquid chromatography, affinity purification protocols (4-8) have been known for a long time. Naturally, electrophoresis can be used just as well to observe molecular or noncovalent interactions of DNA oligomers, provided the complex has distinct electrophoretic properties different from those of the free molecules. Therefore, affinity capillary electrophoresis (ACE) can be a powerful tool for studying DNA-drug or DNA-biopolymer interactions. Several reviews discussing these aspects of ACE have been published in recent years (9-19). The crucial aspects of DNA in this field are covered comprehensively in a recent overview article (20). 

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