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Fig. 7 | Microbial Cell Factories

Fig. 7

From: Protein purification strategies must consider downstream applications and individual biological characteristics

Fig. 7

Workflow of a protein production process. Each protein production process starts with the strategy design. The biochemical characteristics of the protein of interest and the intended downstream applications, as indicated in Table 1 and Fig. 1, need to be considered when deciding which expression host organism to use and how to design the expression construct. After cloning the gene(s) of interest into a suitable expression plasmid, the sequence must be verified. Next, the most optimal expression conditions in the host organism of choice (usually E. coli, yeast, insect, or mammalian cells) are determined. This includes the screening of various parameters such as the expression strain, the expression medium, growth temperature, time, etc. Once the best condition to obtain soluble protein(s) has been found, one can proceed to large-scale protein purification. At this step, it is important to decide on the chromatographic methods that will be used (affinity chromatography, ion exchange, size-exclusion chromatography, …) and to find buffer conditions in which the protein remains in a soluble, properly folded state. Appropriate quality controls throughout the entire process are important to make sure the protein of interest is stable, non-aggregated (Fig. 5), and in a native state. The purified protein can then be used in various downstream applications (Table 1), such as biophysical characterization, interaction studies, structural analysis, immunization, cell assays, etc.

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