Why Spot-Tag can be used to purify any protein
Proteins often have very different individual characteristics and requirements for manipulation. Not every purification tag in combination with the desired purification resin is suitable for every protein. Some proteins are sensitive towards wash or elution buffers, while others are difficult to purify because the selected affinity resin can be contaminated by host cell proteins. Taking these points into consideration, the selection of both the affinity tag and the affinity resin can have a strong impact on protein purity and yield, which is often dependent on the target protein. Requirements for an affinity resin comprise (i) high purity (minimal contaminations), (ii) high yield of the purified protein, and (iii) a broad buffer stability. Ideally, affinity tags should be (iv) inert and (v) short in order to not or minimally affect the purified protein.
An overview of commonly used affinity tags for protein purification can be found here: https://resources.chromotek.com/blog/tags-for-affinity-purification
Spot-tag and Spot-Cap system for protein purification
Spot-Cap® is a novel affinity resin, which binds the 12 amino acid Spot-tag® with high selectivity. Spot-Cap comprises a single domain antibody from alpaca, also called Nanobody or VHH that is conjugated to agarose beads. This resin overcomes limitations of both chemical resins and antibody-based resin because it combines highest selectivity with high binding capacity and effective, mild elution.
Spot-Tag® (sequence PDRVRAVSHWSS) is a flexible peptide-tag of 1.4 kDa size. It preserves native protein folding and full function of the Spot-tagged fusion protein because of its flexible structure. Spot-tag can be fused to the N- or C-terminus, also internal positions can be used. Tag removal may not be necessary after purification because the small, inert Spot-tag is compatible with biochemical downstream assays.
Spot-tag in combination with Spot-Cap are especially suited for the purification of proteins with special requirements as outlined below.
Requirements of special proteins and impact on the selection of the affinity resin for purification
In order to avoid overexpression and possible subsequent generation of inclusion bodies, expression systems that secrete the tagged protein to the cell media are used. Secretion of recombinantly expressed proteins also supports the formation of disulfide bonds and prevents degradation by host cell proteases. The concentration of the secreted tagged protein will be low because of the relatively large volume of the cell media. Hence, a high affinity purification resins is needed to efficiently capture the expressed protein. The application of the His-tag may not be suitable because of the relative low affinity of the Ni/NTA resin. Also, Streptavidin/avidin resin may not be applicable for purification as the cell media often contains biotin. Biotin competes with the Strep- and Strep-Tactin-tagged proteins for binding to streptavidin/avidin resins and thus considerably reduces purification yield. ChromoTek’s Spot-Cap resin has a high affinity to the Spot-Tag and is thus capable to bind Spot-tagged proteins at low concentrations respectively in large volumes. Spot-Cap also has a high selectivity to Spot-tag and doesn’t bind components of the cell medium.
Membrane proteins play an essential role in cellular processes like signal transduction, communication, and energy production to mention a few. Due to their hydrophobic nature, the production of tagged membrane proteins can be challenging. Low expression yields and the presence of detergents, which are needed for solubilization, further complicate their purification. Immunoglobulin-based resins as well as hydrophobic or ion-exchange resins may not be suitable for the purification of membrane proteins, because detergents can compromise the structural integrity and hence the interacting capability. In addition, the purification of low expressed membrane proteins does require a purification resin with high affinity and high elution efficiency. Although immunoglobulin-based resin can bind the purified protein tightly and may be able to capture even lowest amounts of the recombinant protein, these resins usually lack gentle and efficient elution capability. Besides the selection of the purification resin also the position of the purification tag is crucial for the purification of membrane proteins. Peptide tags are typically positioned at the C-terminal side to minimize interference with protein targeting. However, N-terminal and internal positions of the tag must be considered if the peptide tag is covered in the detergent micelle during solubilization and can’t access the purification resin. Spot-Tag can be positioned on the C- and N-terminus and also integration in transmembrane loops is possible. Spot-Cap resin captures and releases the Spot-tagged protein efficiently high concentrations of detergent, i.e. 2% DDM, 2% NP-40, or 2% Triton X-100.
Metalloproteins are a large and diverse protein class and contain e.g. Fe, Cu, or Zn ions. These proteins have many different functions in cells, e.g. protein storage and transport, enzymatic activity, and signal transduction. Metalloprotein purification can be challenging, because complexing reagents or chelating agents like EDTA must be avoided in the binding, wash, and elution buffers as they may form complexes the metal ions and extract metal ions from the protein. In addition, reducing reagents at relative high concentration may have to be added to the buffers to keep the metal ion at the required oxidation state to maintain the protein’s function. Ni/NTA resins may not be used for purification of metalloproteins, because these resins can pull the metal ions from the metalloprotein’s active site leaving only the apo-enzyme behind. Furthermore, these resins may leak their metal ions like Ni and hence may damage the metalloprotein by metal ion replacement. Antibody based purification resins may be sensitive to reducing reagents, because the disulfide bonds of the immunoglobulin may break and disassemble the antibody. In contrast, the Nanobody-based Spot-Cap doesn’t contain metal ions and it can be used with buffers containing reducing reagents like 10 mM DTT, 10 mM ß-mercaptoethanol, and 10 mM TCEP.
Low abundant proteins
For multiple reasons, the expression of a peptide tagged protein can result in a low abundance, e.g. when secreted proteins or membrane proteins are purified (see above). Highly affine purification resins, which have affinities (dissociation constants) in the low nanomolar to picomolar range, must be applied to achieve effective binding. In addition, gentle and efficient elution conditions are needed to elute the bound protein from the resin. Because of its high affinity, the Spot-Cap resin can be used for purification of Spot-tagged proteins at low concentrations and purified proteins can be easily eluted with Spot-peptide.
A protein’s stability, structure, and function can be compromised by multiple factors during purification:
- For purification of sensitive peptide tagged proteins a gentle elution is mandatory, i.e. the competitive elution by free peptide at the lowest peptide concentration possible. This method can result in low yield protein if antibody-based purification resins are used, because the antibody’s high affinity leads to an incomplete elution, which can be even further reduced at low temperatures. The elution of Spot-tagged proteins from Spot-Cap can be conducted with just 0.1 mM Spot-peptide at +4°C and leads to high yield and pure proteins.
- Special buffer components may be needed to solubilize a sensitive tagged protein, which are incompatible with the purification resin. Hence, a broad buffer compatible and chemically stable purification resin may have to be used. Spot-Cap is functional in buffers containing high salt (i.e. 1 M NaCl), chaotropic reagents (i.e. 2 M Urea), reducing reagents (i.e. 10 mM DTT, 10 mM ß-mercaptoethanol, or 10 mM TCEP), and detergents (i.e. 2% DDM, 2% NP-40, or 2% Triton X-100). Binding, wash, and elution buffers can be tailored to the specific protein needs.
- Elevated temperatures can also reduce the tagged protein’s function during purification, and it is generally recommended to conduct protein purification at +4°C. Purification of Spot-tagged proteins with Spot-Cap has been optimized for purification at +4°C temperature.
- The tagged protein may not be stable in the wash/elution buffer. Therefore, a fast one-step purification step should be used to minimize the exposure of the protein to the problematic buffer. The His-Tag and Ni/NTA resins do usually require additional purification steps to yield pure protein, but every additional purification step will decrease the yield of the sensitive protein further. Therefore, high selective and high affine purification resins should be applied. Antibody-based affinity resins like Spot-Cap can be used for one step purifications, which will deliver highly pure proteins after a single purification step because of their high selectivity.
- The peptide tag can also have an impact on the protein’s stability, structure, and function: only inert, flexible peptide tags should be used. Furthermore, it is generally recommended to test C- and N-terminal positions of the tag, possibly also internal positions to overcome interference of the peptide tag. Spot-Tag (sequence PDRVRAVSHWSS) is a flexible, 12 amino acid tag of 1.4 kDa size that does generally not interfere with protein stability and function. For most downstream applications Spot-tag can remain at the purified protein without interference.
For more information on the Spot-Tag click here: https://www.chromotek.com/technology/spot-capture-detection-system/spot-tag/
For more information on the Spot-Cap for protein purification click here: https://www.chromotek.com/products/detail/product-detail/spot-cap/
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