The addition of an incubation step with MgCl 2 and ATP allows the removal of these contaminants ( Figure 1).įast Protein Liquid Chromatography (FPLC) and High Performance Liquid Chromatography (HPLC) are common techniques that depend on expensive instrumentation to generate high yield of the purified protein of interest. Common contaminants in such purification processes are Heat Shock Proteins (HSP). Additionally, the protocol does not require a dialysis step when switching from the GST to the His-purification step column (TALON resin). The small His-tag fused to the other protein extremity serves in a second purification step to increase protein purity by washing off the cleaved GST, as well as degraded proteins and other contaminants. The possibility to cleave off the GST with the PreScission enzyme (with recognition sequence LeuGluValLeuPheGln/GlyPro, resulting in the addition of only two amino acids) has many advantages, for instance, this strategy avoids alterations of the physiological protein functions due to allosteric hindrance. Furthermore, using GST guarantees cost-effectiveness of our method 1. The GST is a long tag (29 kDa), which is highly efficient for purification on glutathione Sepharose beads. Our method involves the fusion of a GST-tag at the N-terminus and a His-tag at the C-terminus of the protein of interest, for an optimal ratio between quantity and purity of the desired protein. In contrast to existing recombinant protein purification protocols in which single tags are used, we established the unique combination of two tags. In recent studies, Flag- or HA-affinity purification has been widely used. Affinity-tag purification strongly increases target-specificity, as in most of the cases the tag will be unique to the protein of interest.
At the same time, the latter chromatography methods demand expensive experimental setup. This problem may be circumvented with the use of multiple purification columns, which is time consuming. The latter protein characteristics are shared by a variety of proteins, which increases considerably the chance of contaminating proteins in conventional protein purification strategies. Conventional ways of protein purification like ion exchange chromatography and size exclusion chromatography rely on physical properties of the target protein such as its isoelectric point and charge or size, respectively. The purification of recombinant proteins is crucial to address fundamental questions in biochemistry.
In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. Additionally, we incorporated MgCl 2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. The method presented here does not require an expensive instrumental setup, such as FPLC. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Hence, it is subsequently cleaved off the protein using the PreScission enzyme 6. However, it might influence physiological properties of the protein. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression 5. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) 2,3 and a C-terminal 10xHis tag 4, which are both fused to the protein of interest.
Protein purification protocols should combine efficiency, simplicity and cost effectiveness 1. Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest.
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