Tandem Affinity Purification (TAP) is a protein purification technique where two consecutive tag genes are molecularly cloned and fused with the target protein gene. Through the expression vector, the fused protein gene is introduced into the target cells, controlling the expression level of the target protein. Under conditions close to physiological states, the cell lysate is subjected to two-step elution using affinity columns that interact with the tags, thereby obtaining a protein purification technique for the target protein and its interacting proteins.
Structure and Components of TAP Tags:
The TAP technique relies on the use of engineered tags that facilitate the purification of target proteins and their associated complexes. These tags consist of specific structural elements and functional components designed to optimize the purification process. Here, we provide a detailed overview of the structure and components of TAP tags:
TAP Tag Structure
TAP tags typically comprise two consecutive tags linked together by cleavage sites recognized by specific proteases. The tandem arrangement of tags allows for sequential affinity purification steps, enhancing the specificity and efficiency of protein purification. Commonly used TAP tags include Protein A, Protein G, polyhistidine (6×His), CBP, SBP, c-Myc, FLAG, and StrepII, each offering distinct advantages in terms of affinity binding properties, elution conditions, and compatibility with downstream applications.
Components of TAP Tags
The components of TAP tags can be broadly categorized into affinity-binding domains, cleavage sites, and linkers:
- Affinity-Binding Domains: Affinity-binding domains are responsible for the specific interaction between the TAP tag and the affinity matrix used for protein purification. These domains, such as Protein A, Protein G, and polyhistidine (6×His), possess high affinity for commonly used chromatographic resins, allowing for efficient capture and immobilization of the tagged protein.
- Cleavage Sites: Cleavage sites within TAP tags serve as recognition sequences for specific proteases, enabling precise removal of the tag following purification. The Tobacco Etch Virus (TEV) protease cleavage site (ENLYFQG) is commonly incorporated into TAP tags due to its high specificity and efficiency in cleaving between tandem tags.
- Linkers: Linkers play a crucial role in connecting the individual components of TAP tags while maintaining their structural integrity and flexibility. These linkers may vary in length, composition, and flexibility to optimize the spatial arrangement of tandem tags and facilitate efficient binding to affinity matrices. Flexible linkers minimize steric hindrance and maximize accessibility of the tags to affinity resins, thereby enhancing purification efficiency.
The modular nature of TAP tags allows for customization and optimization of tag design based on specific experimental requirements and preferences. By selecting appropriate affinity-binding domains, cleavage sites, and linkers, researchers can tailor TAP tags to suit different purification strategies and applications, ranging from the isolation of protein complexes to the characterization of protein-protein interactions and functional studies.
Schematic representation of the tandem-affinity purification strategy (Camp et al., 2012).
Classification of TAP Tags:
TAP tags play a crucial role in the purification of proteins and protein complexes. They are classified based on several criteria, including their position relative to the target protein and the presence of specific cleavage sites. Here, we delve into the classification of TAP tags to better understand their diversity and functionality:
Position Relative to the Target Protein
TAP tags can be positioned either at the N-terminus or the C-terminus of the target protein. This positioning is significant as it can influence the accessibility of the tag to affinity matrices and the subsequent purification efficiency. Traditionally, TAP tags have been predominantly C-terminal due to their ease of construction and minimal interference with protein function. However, the use of N-terminal tags is gaining popularity, particularly in cases where C-terminal tagging may interfere with protein folding or function.
Presence of TEV Protease Cleavage Sites
Another important classification criterion for TAP tags is the presence of specific cleavage sites, notably those recognized by the Tobacco Etch Virus (TEV) protease. TEV cleavage sites offer a convenient means of removing the tag post-purification, leaving behind the native protein sequence. TAP tags may contain one or more TEV cleavage sites strategically positioned between the tandem tags, allowing for precise cleavage and tag removal. Tags lacking TEV cleavage sites require alternative methods for tag removal, which may involve harsher conditions and could potentially affect protein integrity.
Diversity of TAP Tags
TAP tags exhibit considerable diversity in terms of their composition and properties. Commonly used tags for the first affinity purification step include Protein A, Protein G, and polyhistidine (6×His), each offering distinct advantages in terms of affinity binding and purification efficiency. For the second affinity purification step, a variety of tags such as CBP, SBP, c-Myc, FLAG, and StrepII are utilized, each characterized by unique properties such as mild elution conditions and minimal interference with protein structure and function. The choice of TAP tags depends on factors such as the nature of the target protein, the intended downstream applications, and the compatibility with purification protocols.
Optimization Strategies of TAP Tags
Codon Optimization
Codon optimization involves redesigning the nucleotide sequences encoding TAP tags to better match the codon usage preferences of the host organism or cell line. This optimization ensures efficient translation and expression of the tagged protein in the target system, thereby maximizing protein yield and enhancing purification efficiency. By aligning the codon usage with the cellular machinery, codon optimization minimizes translation errors and reduces the risk of protein misfolding or degradation.
Structural Modifications
Structural modifications of TAP tags aim to improve their affinity binding properties, minimize non-specific interactions, and enhance overall purification efficiency. These modifications may include altering the amino acid composition, introducing specific motifs or linkers to optimize tag accessibility, or engineering additional affinity-binding domains to enhance tag-protein interactions. Structural modifications also involve fine-tuning the length and flexibility of linkers between tandem tags to optimize the spatial orientation and accessibility of the tags during purification.
Development of Novel Tags
Continuous innovation in TAP tag design involves the development of novel tag sequences with improved properties and functionalities. Novel tags may be engineered to exhibit enhanced affinity binding to specific affinity matrices, increased stability under various purification conditions, or enhanced resistance to proteolytic degradation. Additionally, novel tags may incorporate innovative features such as inducible cleavage sites, fluorescent or affinity tags for real-time monitoring or downstream applications, or cell-penetrating peptides for intracellular delivery of tagged proteins.
Multi-Tagging Strategies
Multi-tagging strategies involve the incorporation of multiple affinity tags or purification modules within a single TAP tag construct. By combining different affinity tags with complementary properties, such as high affinity binding, mild elution conditions, or specific recognition sequences for proteolytic cleavage, multi-tagging strategies offer enhanced flexibility and robustness in protein purification. Multi-tagging also enables parallel or sequential purification steps, allowing for the isolation of protein complexes with high purity and yield.
Optimization of Purification Protocols
In addition to optimizing tag sequences, purification protocols can be optimized to maximize the efficiency and yield of TAP-based protein purification. Optimization of elution conditions, buffer compositions, and chromatographic parameters can significantly impact the purity, recovery, and stability of purified protein complexes. Furthermore, the development of streamlined purification protocols with reduced steps and simplified procedures can minimize sample loss and increase throughput, making TAP-based purification more accessible and scalable for high-throughput applications.
Trends in TAP Tag Development
TAP has matured continuously and is currently utilized in yeast, plant cells, and mammalian cells. However, while the use of TAP tags has reduced non-specific binding proteins, it has also increased the steps involved in protein purification, resulting in lower protein recovery rates and restricting the application scope of TAP. Particularly for proteins with low expression levels in their native state, purification using TAP tags requires a large number of cells, posing a bottleneck in current TAP applications. Discrepancies between the expression levels of exogenous and native proteins, as well as competition from endogenous proteins, present challenges for the TAP system.
To address these issues, optimization of tag codons can be employed to adapt to the codon preferences of different cells, reducing occurrences of low expression or non-expression of exogenous proteins caused by tag proteins. Furthermore, optimization of tag protein structure and the development of new tags can minimize the non-specific binding of proteins to tags, thereby enhancing the affinity of tag proteins for affinity columns. Efforts can also focus on optimizing processes and simplifying separation steps to minimize protein loss and increase the recovery rate of target proteins. In conclusion, with the continuous improvement of TAP tags, TAP will play an increasingly important role in the study of protein-protein interactions.
Reference
- Camp, Nathan Daniel. WTX is a novel regulator of ubiquitination in the Wnt/β-catenin and KEAP1/NRF2 pathways. University of Washington, 2012.
