Detection and prevention of protein aggregation before, during, and after purification.
The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.
Bondos
Sarah E
SE
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA. bondos@bioc.rice.edu
Bicknell
Alicia
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D015153:Blotting, Western; D002021:Buffers; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification; D018398:Homeodomain Proteins / Q000737:chemistry / Q000302:isolation & purification; D056968:Lac Repressors; D009154:Mutation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D015534:Trans-Activators / Q000737:chemistry / Q000302:isolation & purification; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification
C082932:ATXA protein, Bacillus anthracis; D001426:Bacterial Proteins; D002021:Buffers; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D018398:Homeodomain Proteins; D056968:Lac Repressors; D011506:Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C066177:Ubx protein, Drosophila