8 results on '"RYAN, BARRY J."'
Search Results
2. Avoiding Proteolysis During Protein Chromatography
- Author
-
Ryan, Barry J., primary
- Published
- 2010
- Full Text
- View/download PDF
3. Approaches to Avoid Proteolysis During Protein Expression and Purification.
- Author
-
Henehan GT, Ryan BJ, and Kinsella GK
- Subjects
- Proteolysis, Amino Acids, Cell Death, Peptide Hydrolases, Endopeptidases
- Abstract
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 proteases before they are physically separated from the protein of interest via column chromatography. In this chapter, commonly used approaches to reducing or avoiding proteolysis during protein expression and purification are reviewed., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
- Full Text
- View/download PDF
4. Protein Extraction and Purification by Differential Solubilization.
- Author
-
Ryan BJ, Kinsella GK, and Henehan GT
- Subjects
- Cell Death, Chromatography, Affinity, Chromatography, Gel, Chromatography, Ion Exchange, Computational Biology
- Abstract
The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this process. Selectively altering the solubility of a protein is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of a polypeptide. Precipitation of proteins, released from cells upon lysis, is often used to concentrate a protein of interest before further purification steps (e.g., ion exchange chromatography, size exclusion chromatography etc).Recombinant proteins may be expressed in host cells as insoluble inclusion bodies due to various influences during overexpression. Such inclusion bodies can often be solubilized to be reconstituted as functional, correctly folded proteins.In this chapter, we examine strategies for extraction/precipitation/solubilization of proteins for protein purification. We also present bioinformatic tools to aid in understanding a protein's propensity to aggregate/solubilize that will be a useful starting point for the development of protein extraction, precipitation, and selective re-solubilization procedures., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
- Full Text
- View/download PDF
5. Differential Precipitation and Solubilization of Proteins.
- Author
-
Ryan BJ and Kinsella GK
- Subjects
- Computational Biology methods, Databases, Protein, Protein Folding, Recombinant Proteins chemistry, Solubility, Web Browser, Chemical Precipitation, Proteins chemistry
- Abstract
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 proteins. Here, appropriate bioinformatics tools to aid in understanding a protein's propensity to aggregate and solubilize are explored as a backdrop for a typical protein extraction, precipitation, and selective resolubilization procedure, based on a recombinantly expressed protein.
- Published
- 2017
- Full Text
- View/download PDF
6. Avoiding Proteolysis During Protein Purification.
- Author
-
Ryan BJ and Henehan GT
- Subjects
- Chromatography methods, Hydrolysis drug effects, Peptide Hydrolases chemistry, Peptide Hydrolases metabolism, Protease Inhibitors pharmacology, Proteins metabolism, Proteolysis drug effects, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Proteins chemistry, Proteins isolation & purification
- Abstract
All cells contain proteases which hydrolyze the peptide bonds between amino acids in 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; firstly inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographical purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing or avoiding proteolysis during protein purification and subsequent chromatography are reviewed.
- Published
- 2017
- Full Text
- View/download PDF
7. Avoiding proteolysis during protein chromatography.
- Author
-
Ryan BJ
- Subjects
- Amino Acid Sequence, Animals, Humans, Peptide Hydrolases metabolism, Protease Inhibitors pharmacology, Proteins chemistry, Chromatography methods, Proteins isolation & purification, Proteins metabolism
- Abstract
All cells contain proteases, which effect catalytic hydrolysis of the peptide bond between amino acids in the protein backbone. Typically, proteinases are prevented from nonspecific proteolysis by regulation and physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis to release a protein of interest. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; first the inhibition of proteolysis in situ, followed by the separation of the protease from the protein of interest via chromatographical purification. Proteinase inhibitors are routinely used to limit the effect of the proteinases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing proteolysis during chromatography are reviewed.
- Published
- 2011
- Full Text
- View/download PDF
8. Differential precipitation and solubilization of proteins.
- Author
-
Ryan BJ
- Subjects
- Ammonium Sulfate chemistry, Protein Refolding, Proteins genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Solubility, Trichloroacetic Acid chemistry, Chemical Fractionation methods, Chemical Precipitation, Proteins chemistry, Proteins isolation & purification
- Abstract
Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physico-chemical 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 over-expression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilised so as to reform functional, correctly folded proteins. Here, a typical protein extraction, precipitation, and selective resolubilisation procedure is outlined, based on a recombinantly expressed protein.
- Published
- 2011
- Full Text
- View/download PDF
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