| Abstrakt: |
There are a variety of reliable methods for cellular disintegration and extraction of proteins ranging from enzymatic digestion and osmotic shock to ultrasonication, and pressure disruption. Each method has inherent advantages and disadvantages. Generally vigorous mechanical treatments reduce extract viscosity but can result in the inactivation of labile proteins by heat or oxidation, while gentle treatments may not release the target protein from the cells, and resulting extracts are extremely viscous. Depending on the cell type selected as the source for target protein expression, cellular extracts contain large amounts of nucleic acid, ribosomal material, lipids, dispersed cell wall polysaccharide, carbohydrates, chitin, small molecules, and thousands of unwanted proteins. Isolation and recovery of a single protein from this complex mixture of macromolecules presents considerable challenges. The first and possibly most important of these challenges is generation of a cellular extract that can be efficiently manipulated in downstream processes without inactivation or degradation of labile protein targets. Cell disruption techniques must rapidly and efficiently lyse cells to extract proteins with minimal proteolysis or oxidation while reducing extract viscosity caused by cell debris and genomic DNA contamination. Advanced bioprocessing equipment and reagents have been developed over the past twenty years to complement established disruption procedures and accomplish these tasks with even greater success. This chapter will summarize these advances and describe detailed protocols for some of the most popular methods for protein extraction. |
