Purification strategies

Cells contain thousands of different proteins. A major problem for protein chemists is to purify a chosen protein so that they can study its specific properties in the absence of other proteins. Proteins have been separated and purified on the basis of their two prominent physical properties size and electrical charge. A more direct approach is to employ affinity purification strategies that take advantage of the biological function or similar specific recognition properties of a protein (see Table 5.5 and Chapter Appendix). 

The purification strategy for CBPs is conceptually simple. Proteins from carotenoid-rich tissues are separated under nondenaturing and relatively aqueous conditions where carotenoids are expected to remain bound to the CBPs. CBPs are then detected by the color of the carotenoids. 

Spider silk proteins from plants remain soluble at high temperatures, allowing them to be enriched by boiling [26]. In order to enrich the spider silk-ELP fusion protein, we therefore exposed tobacco leaf extracts to heat treatment at 95°C for 60 min and then cleared the supernatant by centrifugation. In further steps, the reversible precipitation behavior of ELP fusion proteins was exploited to develop a suitable purification strategy. For the selective precipitation of SOl-lOOxELP, NaCl was added at a final concentration of 2 M and the temperature was increased to 60 °C. In this man-... 

Figure 11.11 Overview of the procedure by which hCG may be purified from the urine of pregnant females at laboratory scale. Production-scale systems would be at least partially based upon such a purification strategy. Although initial concentration steps could involve precipitation, the use of ultrafiltration would now be more common...

Expression of a recombinant protein using an inducible vector system would permit expression at endogenous levels to simulate physiologic levels of expression of a protein of interest. Tandem affinity purification strategies have recently been employed and facilitate the analyses of highly interactive proteins when the bait protein is expressed at endogenous levels. Immunoaffinity or immunoprecipitation followed by LC-MS/MS does not readily permit determination of the stoichiometry of interacting partners. Additionally, when compared to yeast hybrid experiments, it is difficult to determine whether interactions are binary when identified in complexes by MS/MS. 

Following a similar strategy, an ingenious mixed resin bed quench and purification strategy was devised for the Dess-Martin periodinane mediated conversion of alcohols to carbonyls. This hypervalent iodine oxidant was viewed as containing an inherent masked carboxylic acid functionality that was revealed at the end of the reaction (Species (11) Scheme 2.30). Therefore purification was easily achieved by treatment of the reaction mixture with a mixed-resin bed containing both a thiosulfate resin and a polymeric base. The thiosulfate polymer was used to reduce excess hypervalent iodine lodine(V) and (III) oxidation states species to 2-iodoben-zoic acid (11), which was in turn scavenged by the polymeric base [51]. 

Scheme 2.31 Deprotection and purification strategy based on scavenger technology ...

The introduction of these alternative approaches has permitted the direct trans-ferral and smooth integration of many versatile solution phase reagents from mainstream chemistry catalogues straight into practical protocols for use in com-hinatorial parallel hbrary generation thus broadening the amenable chemistry base. As with aU procedures it is the flexibihty and synthetic scope provided by these simple operations that has enhanced their utility as alternative reaction conditions and purification strategies. 

A very different purification strategy was initiated by Valdemoro et al. [71] in 2003 and extended by Alcoba and Valdemoro [72] in 2005. Thus these authors... 

From the begining of the development of the RDM theory the need to render N-and 5-representable a 2-RDM obtained by an approximative method was patent. The development first of the spin-adapted reduced Hamiltonian methodology and, more recently, that of the second-order contracted Schrodinger equation rendered the solution of this problem urgent. The purification strategies... 

Another extension of this theoretical smdy is the consideration of both an economical and an effective purification strategy for the 4-RDM. The need for such a purification scheme is motivated by the need to have an N- and 5-representable 4-RDM if one wishes to solve the fourth-order modified contracted Schrodinger equation [62, 64, 87]. There have already been several attemps to purify both the 3-RDM and 4-RDM [18, 34, 52]. In particular, a set of inequalities that bound the diagonal and off-diagonal elements of these high-order matrices have been reported [18]. However, the results obtained with this approach within the framework of the fourth-order modified contracted Schrodinger equation (and the second-order contracted Schrodinger equation) were not fully satisfactory because the different spin-blocks of the matrices did not appear to be properly balanced [87, 114]. 

Figure 2.3 Schematic representation of the workflow for automated protein purification strategies. ( offline tag cleavage involves a manual step.)...

A number of purification strategies are in place that have been tailored for specific proteins derived from particular host production platforms. For example, downstream protein extraction from seeds is quite simple... 

A combination of these optimized tools are used for large-scale operations essential to obtaining sufficient quantities of recombinant protein for preclinical and clinical development. Additional scale-up and purification strategies are discussed in the following section. Regardless of the purifi-... 

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