RESUMO
Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.
Assuntos
Bioquímica/métodos , Proteínas/isolamento & purificação , Bioquímica/instrumentação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/isolamento & purificação , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Intrinsically disordered proteins are anticipated to be more prone to aggregation than folded, stable proteins. Chemical additives included in the buffer can help maintain proteins in a soluble, monomeric state. However, the array of chemicals that impact protein solubility is staggering, precluding iterative testing of chemical conditions during purification. Herein, we describe a filter-based aggregation assay to rapidly identify chemical additives that maintain solubility for a protein of interest. A hierarchical approach to buffer selection is provided, in which the type of chemical which best improves solubility is first determined, followed by identifying the optimal chemical and its most effective concentration. Finally, combinations of chemical additives can be assessed if necessary. Although this assay can be applied to purified protein, partially purified protein, or aggregated protein, this protocol specifically details the use of this assay for crude cell lysate. This approach allows identification of solubility-promoting buffers prior to the initial protein purification.