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Full-Text Articles in Molecular Biology
Approaches To Avoid Proteolysis During Protein Expression And Purification, Gary T. Henehan, Barry J. Ryan, Gemma K. Kinsella
Approaches To Avoid Proteolysis During Protein Expression And Purification, Gary T. Henehan, Barry J. Ryan, Gemma K. Kinsella
Books/Book Chapters/ Proceedings
All cells contain proteases, which hydrolyze the peptide bonds between amino acids of a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach: first, inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographic purification. Protease inhibitors are routinely used to limit the effect of the …
Differential Precipitation And Solubilisation Of Proteins, Barry J. Ryan, Gemma K. Kinsella
Differential Precipitation And Solubilisation Of Proteins, Barry J. Ryan, Gemma K. Kinsella
Books/Book Chapters/ Proceedings
Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilized so as to reform functional, correctly folded …