Proteins purification, requirements

Inert systems are used for two reasons. Purification of proteins can be contaminated and enzymes can be deactivated with metals ions extracted out of stainless steel. Also, inert systems are resistant to concentrated salt solutions. Some protein purifications require in excess of 150 mM salt. 

Protein crystallography often requires special constructs or mutants to facilitate crystallization it also requires large quantities of highly purified protein. Thus to move forward in a timely fashion, it is important that an industrial structural biology group employ molecular biologists and individuals with expertise in protein purification. 

Compared with isolated enzymes, application of whole cells as biocatalysts is usually more economical since there is no protein purification process involved. Whole cells can be used directly in chemical processes, thereby greatly minimizing formulation costs. Whole cells are cheap to produce and no prior knowledge of genetic details is required. Microorganisms have adapted to the natural environment and produce both simple and complex metabolic products from their nutrient sources through complex, integrated pathways. 

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. 

A major feature of HTP protein production pipelines is the inclusion of a screening step at a relatively small scale to identify constructs suitable in terms of soluble protein yield for scale-up and subsequent protein purification. Given the relatively high cost of the latter steps in terms of time and resources, particularly if eukaryotic expression is required, the screening stage is seen as crucial to the overall process. In this section the approaches to the evaluation of expression at small scale will be reviewed. 

All purification procedures require a method for quantifying or assaying the protein of interest in the presence of other proteins. Purification can be monitored by assaying specific activity. 

Purification protocols should always aim for brevity, to minimize complexity and cost. The goal of the protein purification procedure, besides a pure protein, is a purification table listing all the operations undertaken, with results on overall yield, specific activity, and purification factor. An assay for both protein function and protein concentration is required at every step. 

Fortuitous ligands. Ligands from the expression host (e.g. fatty acids from Escherichia coll) could be noncovalently bound, e.g. in the active site of the overexpressed target protein. This is a serious, but rare, problem and may require an additional refolding step in the protein purification protocol. The presence of any noncovalently bound ligands in the target protein can be checked by nondenaturing ESI-MS. 

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