Affinity-Tag Mediated Capture for Label-Free Biomolecular Interaction Analysis
In the post-genomic era, most analytical tools for studying the structure and function of recombinant proteins are limited by being specific to each individual protein, its sequence, and conformation. This narrow focus is often a bottleneck, limiting the number of proteins that can be processed or analyzed. Broader methods for detection that are applicable to all protein expression systems can streamline the development and analysis of a wide range of recombinant proteins in a high-throughput process.
Affinity tags are a polypeptide amino acid sequence that can be engineered at the N- or C-terminus of an expressed protein to facilitate more generalized detection and purification. Tagged fusion proteins are designed for their affinity to a binding partner that is independent of the recombinant protein. Affinity-tagged fusion proteins allow for capture of the recombinant proteins without changing their unique physiological properties, tertiary structure, and biological activity.
There are a variety of affinity tag-protein strategies, and the use of affinity tags depends on the target protein, the expression system, and the application. The Octet QK and Octet RED Systems provide an easy-to-use, label-free high-throughput system to capture affinity-tagged target proteins for quantitative or kinetic molecular interaction analysis. Table 1 lists a variety of affinity tags that have been used successfully on the Octet QK and Octet RED Systems.
The polyhistidine-tag, known as the His6-tag, is an amino acid sequence consisting of six histidine residues. It exhibits a strong interaction to immobilized metal ion matrices, such as Ni-NTA. Label-free analysis for His6-tagged proteins can be developed using either the Octet QK or Octet RED instruments and streptavidin biosensors.
By taking advantage of the flexible design of the biosensor tray, nickel-activated Biotin-NTA or an anti-His directed antibody can be batch-immobilized on streptavidin biosensors. These ligand-coated biosensors can then be used with the Octet QK or Octet RED instruments to capture His-tagged proteins from a crude sample for further analysis.
Example 1: Binding Antibodies to Antigens
The binding of antibodies to antigens was assessed by using the Octet RED System to conduct kinetic screening. Biotin-Ni NTA-coated Streptavidin High Binding biosensors were batch-immobilized offline (i.e., outside the Octet instrument). On the Octet RED System, a His-tagged antigen was immobilized onto the coated biosensors. A brief PBS wash ensured the stability of the immobilization. Subsequent analysis using various antibodies at a 1:20 dilution provided association and dissociation profiles for the antibodies’ binding to the antigen. As shown in Figure 1, successful capture of the His-tagged antigen enabled screening for antibody binding and rank ordering.
Example 2: Comparison of Strategies for Evaluating His-Tagged Protein 1
The specific protein:protein interactions determine which strategy is optimal for capturing a His-tagged protein. Two such methods were evaluated, comparing biotinylated anti-His antibody (online immobilization) with biotinylated Ni-NTA (offline immobilization) as shown in Figure 2. A post-immobilization baseline (BL) established a stable surface for capturing His-tagged Protein 1.
Each capture surface was assayed with and without crosslinking to assess the potential for conferring enhanced stability for extended kinetic analysis with Protein 2. As shown in Figure 2, the Biotin-Ni NTA surface produced results that were similar to previously obtained results. Additionally, crosslinking did not have any effect and was not necessary for kinetic analysis.
The FLAG-tag system is based on an eight amino acid sequence that is recognized by an anti-FLAG antibody. On the Octet QK System, an anti-FLAG antibody was immobilized onto Amine Reactive Biosensors online (20 mg/mL for 12 minutes). A recombinant FLAG protein was immobilized for 15 minutes. A five-minute baseline ensured a stable surface for subsequent association of the 70 kDa receptor for 15 minutes. Dissociation of the FLAG protein:receptor interaction was monitored for 15 minutes. As the data in Figure 3 illustrates, FLAG-tag mediated capture was achieved without interfering with the recognition of the FLAG-protein:receptor interaction.
The GST-tag is large protein tag (220 amino acids) that is recognized by glutathione. The use of GST as a tag helps to protect the expressed target protein from intercellular protease cleavage, thereby enhancing its stability.
On the Octet QK System, biotinylated glutathione was immobilized onto Streptavidin biosensors. After a short wash, GST-BAK1 was captured onto the surface. After another wash, association of FLAG-BRI1 was monitored for approximately 56 minutes and the subsequent dissociation was monitored for approximately 33 minutes. Two different strategies to immobilize different capture surfaces for GST-BAK1 yielded similar affinities (see Figure 4 and Table 2). Additionally, reversing the interaction also produced an affinity that was in good agreement. All three experimental designs were completed in a single day on the Octet QK System.
The Strep-tag II is a small (eight amino acid) sequence that is recognized by streptactin, enabling its purification and analysis under physiological conditions with yields greater than 99% purity. Strep-tag II has 100 times higher affinity than the native form and is more commonly used. Using the Octet QK System, a biotinylated anti-Strep II antibody was immobilized using Streptavidin biosensors. Strep II-tagged protein was immobilized on the biosensors, then a baseline established to confer a stable surface. The immobilized Strep II-tagged protein provided a means to screen for potential receptor binding partners (Figure 5).
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