Protein purification strategies

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

PHA bead-based fusion protein purification strategies have utilized fusions of the target protein to proteins naturally associated with PHA inclusions such as phasins, regulatory, or synthase proteins. Banki et al. [74] used a phasin-intein target protein fusion to anchor the fusion protein to the PHA granule as it forms... 

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-... 

Hedhammar, M., Graslund, T., and Hober, S. 2005. Protein engineering strategies for selective protein purification. Chemical Engineering and Technology 28, 1315-1325. 

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. 

Why Purify Enzymes , 182, 1 strategies and considerations for protein purifications, 182, 9 rethinking your purification procedure, 182, 779. 

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-... 

S. -H. (1996) Strategies for Protein Purification and Characterization, Cold Spring Harbor Lab. Press, Cold Spring Harbor, New York... 

A purification strategy for any protein at any scale of operation can be broadly divided into three sequential stages—capture, intermediate purification, and polishing. Each stage represents a set of specific problems that may be encountered during a purification process. The nature of the sample and the scale of operation will dictate what equipment and methodology are appropriate to solve the problem. Many of the procedures discussed here can be found in Coligan et al. (2001). 

---