KMD Bioscience Co., Ltd.
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2024-06-03 Hits(139)
Protein Production
Tagged proteins refer to proteins that have been genetically modified to include an additional peptide or protein sequence, known as a tag, fused to the target protein. The tag serves various purposes in experimental and research areas.
There are several commonly used affinity tags, including polyhistidine (His-tag), glutathione S-transferase (GST), maltose-binding protein (MBP), and Strep-tag. Each tag has its characteristics and advantages, such as high affinity for specific purification resins or compatibility with particular detection methods.
Protein Localization and Tracking
Some tags, such as green fluorescent protein (GFP), enable the visualization and tracking of the tagged protein within cells or tissues. GFP and other fluorescent tags can study protein localization, movement, and dynamics in real-time, providing valuable insights into cellular processes.
Protein Detection and Purification
Tags are commonly used to facilitate the detection and purification of the tagged protein. The tag provides a specific epitope that can be recognized by antibodies or affinity purification methods.
Protein-Protein Interaction Studies
Tags can be used to monitor protein expression levels and assist in protein engineering and characterization by introducing tags at specific positions in the protein sequence. Proteins of interest can be fused with bait tags and can be selectively separated from interacting proteins using techniques such as co-immunoprecipitation or pull-down analysis.
Tagged Proteins Expression Advantages and Disadvantages
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Advantages |
Disadvantages |
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Simplified Purification |
Tags such as His-tag, GST, and FLAG facilitate easy and efficient purification using affinity chromatography, reducing the number of purification steps required. Simple purification is possible using AC (Adenylate cyclase). Generic two-step purification protocols can often be set up for lab-scale protein production platforms. |
Potential Interference |
Tags can sometimes interfere with the protein’s natural function, structure, or interactions, leading to misfolding or altered activity. Although tags can often be removed via proteolytic cleavage, this additional step can be incomplete or inefficient, and it may leave residual sequences that still affect the protein's function |
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Enhanced Detection |
Tags such as GFP (Green Fluorescent Protein) or epitope tags like FLAG and Myc allow easy detection and quantification of proteins using standard laboratory techniques like Western blotting, ELISA, or fluorescence microscopy. Detection of the tag instead of the target protein moiety allows for a generic detection method in, e.g., protein production platforms for structural biology. |
Immunogenicity |
tags can be immunogenic, potentially leading to immune responses when used in vivo |
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Increased Solubility |
Solubility and stability can be improved. Tags like MBP (Maltose-binding protein) and GST can increase the solubility of the target protein, reducing aggregation and enhancing yield. Targeting information can be incorporated into a tag. A marker for expression is provided. Some tags allow strong binding to chromatography media in the presence of denaturants, making on-column refolding possible. |
Cost and Complexity |
The need for specific reagents and affinity columns for tag-based purification can increase costs and complexity |
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High Purity |
Tagged proteins often achieve higher purity levels, as specific affinity columns can selectively bind the tagged proteins and wash away impurities.
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Cannot completely remove labels |
If the tag needs to be removed, cleavage may not always be achieved at 100%, and sometimes amino acids may be left. (The effectiveness of proteases used for cleavage may be decreased by substances, for example, detergents, in the protein preparation or by inappropriate conditions.) |
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