Protein equipment requirements

Producing the kilograms of tPA necessary to satisfy the world s therapeutic needs requires the special skills possessed by modern biochemical engineers. Sophisticated engineering of the fermentation vessels, culturing conditions, and media compositions is required to culture thousands of liters of mammalian cells. In addition, new extremes of purity must be achieved in order to assure the safety of proteins derived from mammalian cells. The cost of the starting materials and the capacity constraints of the present-day equipment require that yields from each fermentation batch be as high as possible. 

The measurement of fluorescence life-times has great value for probing structural features of proteins. It requires expensive equipment since very rapid extinction of the exciting nanosecond pulse is necessary and the rapid decay of the emission must then be measured. The decay of the tryptophan fluorescence of LADH is biphasic with x = 3.9 and 7.2 ns and these are assigned to buried Trp-314 and exposed Trp-15, respectively. ... 

Polishing. This last process step prepares the product for final formulation or for actual sale. It is designed to remove any aggregated protein, remove residual chromatographic eluent(s), and place the product into a specific solvent. These requirements are admirably served by gel filtration. At this point, the sample volume is small and the product fraction to be applied is fairly clean. The gel and column equipment requirements are now within reason and, the clean samples result in much longer gel life. 

Plasmid purification methods vary in the time, expense, and equipment required and in the purity of the plasmid produced. Purity is important to generate high levels of transfection in a reproducible manner. Potential contaminants include bacterial genomic DNA, RNA, protein, endotoxin, chemical residues, trace metals, and undesirable counterions (159). Depending on the user s endpoint, concern regarding any of these contaminants will vary. For example, if the plasmid product is intended for human clinical trials, strict quality control over of all these factors, among others, must be addressed. Lesser concern is warranted for... 

Among the analytical techniques described in previous sections, ELISA was found to be particularly suitable for routine detection of soy as an allergen in food products. Since commercial kits contain all the materials needed for test execution and the equipment required is comparatively cheap, the assay could also be carried out in unspeciaUzed facilities. The analytical procedure is quite easy and fast, so it could be useful for a quick check of possible contamination or residues from soy. The main disadvantage is that industrial processing could destroy protein structure, and for this reason results related to processed foods should be regarded as presumptive only, never as absolute. 

Methods based on liquid chromatography-mass spectrometry (LC-MS) and universally accepted search algorithms permit reliable identifications of low levels of proteins at high sensitivity [6]. Even semispecialized protein chemistry labs can readily identify proteins at the level of a few picomoles (10 pmol of a 50-kDa protein is 500 ng). Specialized groups with access to the latest advances in HPLC and mass spectrometry routinely work with subpicomolar quantities. Chemical proteomics as discussed here requires the more advanced equipment. 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

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