Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Industrial Protein Synthesis Using Recombinant Microorganisms
Downstream Processing

Following Cell Disruption, debris is removed either by low-speed centrifugation of large volumes or by membrane microfiltration. The protein product is precipitated from the crude or clarified lysate using organic Solvents (alcohol or acetone) or ammonium sulfate. This achieves a 2- to 5-fold enrichment. Unfortunately, the high cost of precipitating agents can significantly increase overall process costs. Alternatively, tangential flow ultrafiltration through membranes with a smaller average pore size than those used for cell concentration or debris removal can be employed to concentrate and isolate total Proteins (Fig. 16.7, B). Although this approach is still under development, it is already clear that it is suitable for Processing volumes ranging from one to several thousand liters; the process can run continuously (which helps reduce equipment footprint) and yield a 10- to 100-fold enrichment (depending on the size and Properties of the target protein).

The required purity level of the protein product depends on its intended application. In some cases, a relatively crude preparation is sufficient, while in others (for example, therapeutic proteins used in medicine), an extremely high degree of purity is required.

Some proteins overexpressed in Cells form insoluble particles known as inclusion bodies. Following cell disruption, they can be easily separated from most other cellular components. Initially, researchers struggled to solubilize the isolated inclusion bodies without causing irreversible Protein Denaturation, but Methods were later developed to refold the recombinant protein and restore its activity. Clearly, all these additional steps increase the cost of the purification process.

Protein solubilization

In some cases, overexpression of recombinant proteins yields both soluble and insoluble products, which complicates the purification process. For example, when the human Insulin-like growth factor I (IGF-I) Gene (molecular weight 7.6 kDa) is expressed in E. coli, approximately 90% of the recombinant molecules localize to the periplasm, while 10% are secreted. To isolate both the soluble and insoluble forms of IGF-I from the periplasm, urea and dithiothreitol were added to high concentrations at alkaline pH for in situ solubilization of the insoluble form. This killed the cells without lysing them, leaving cytoplasmic proteins trapped inside. This resulted in a highly viscous solution, making it difficult to pellet the cells and debris by centrifugation. To address this issue, an aqueous two-phase extraction Procedure was developed to separate the soluble and insoluble products. Both in situ solubilization and aqueous two-phase extraction are highly efficient, enabling the recovery of 80% to 95% of IGF-I from culture volumes ranging from 10 to 1000 L.