Difference between revisions of "Part:BBa K3843005"
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Streptavidin is a highly useful protein in molecular biology due to its high-affinity non-covalent interaction with biotin, a small molecule. This makes the biotin-streptavidin duo valuable as an affinity tag system. However, overexpression of streptavidin can be complicated by its tetrameric nature, making it harder to obtain large amounts for use in diagnostic assays. In a similar sense, the tetrameric nature of streptavidin makes it a bulky and non-optimal protein for use in a translational fusion protein. This is because bulkier fusion proteins tend to exhibit a higher risk of aggregation and instability, which is accentuated by the presence of multiple subunits. To mitigate these complications of working with streptavidin, __________ engineered a monomeric streptavidin using rational protein design. This engineered monomeric streptavidin (henceforth referred to as mSA2) eliminates the complications mentioned above, but displays a markedly lower affinity for biotin than its tetrameric counterpart (CITE). The existing part for monomeric streptavidin is BBa_K1896000. | Streptavidin is a highly useful protein in molecular biology due to its high-affinity non-covalent interaction with biotin, a small molecule. This makes the biotin-streptavidin duo valuable as an affinity tag system. However, overexpression of streptavidin can be complicated by its tetrameric nature, making it harder to obtain large amounts for use in diagnostic assays. In a similar sense, the tetrameric nature of streptavidin makes it a bulky and non-optimal protein for use in a translational fusion protein. This is because bulkier fusion proteins tend to exhibit a higher risk of aggregation and instability, which is accentuated by the presence of multiple subunits. To mitigate these complications of working with streptavidin, __________ engineered a monomeric streptavidin using rational protein design. This engineered monomeric streptavidin (henceforth referred to as mSA2) eliminates the complications mentioned above, but displays a markedly lower affinity for biotin than its tetrameric counterpart (CITE). The existing part for monomeric streptavidin is BBa_K1896000. | ||
| − | This part is an improvement of BBa_K1896000. In hoping to further improve mSA2’s binding affinity to biotin for use in a diagnostic microfluidic assay, Waterloo iGEM (2021) used a computational workflow to perform rational protein design on mSA2. Each amino acid of mSA2 was iteratively mutated using Rosetta to determine the mutations yielding the lowest computational energy scores for mSA2, indicating the greatest stability. Only mutations occurring in the binding site were ultimately considered, as any other mutations were unlikely to affect binding affinity. Then, ligand docking was performed using AutoDock Vina to assess whether the mutations improved binding affinity between | + | This part is an improvement of BBa_K1896000. In hoping to further improve mSA2’s binding affinity to biotin for use in a diagnostic microfluidic assay, Waterloo iGEM (2021) used a computational workflow to perform rational protein design on mSA2. Each amino acid of mSA2 was iteratively mutated using Rosetta to determine the mutations yielding the lowest computational energy scores for mSA2, indicating the greatest stability. Only mutations occurring in the binding site were ultimately considered, as any other mutations were unlikely to affect binding affinity. Then, ligand docking was performed using AutoDock Vina to assess whether the mutations improved binding affinity between mSA2 and biotin; a lower complex energy corresponded to a higher binding affinity. |
| − | Ultimately, | + | Ultimately, three binding site mutations were implemented: T74C, N12A, and Y52F. These mutations were able to decrease the energy of the mSA2-biotin complex by _______ kcal/mol compared to the unmutated mSA2. The following image depicts the improved mSA2, with the mutated residues highlighted: |
| + | |||
| + | IMAGE LINK | ||
Revision as of 06:59, 18 October 2021
Affinity-improved monomeric streptavidin (mSA2+)
Streptavidin is a highly useful protein in molecular biology due to its high-affinity non-covalent interaction with biotin, a small molecule. This makes the biotin-streptavidin duo valuable as an affinity tag system. However, overexpression of streptavidin can be complicated by its tetrameric nature, making it harder to obtain large amounts for use in diagnostic assays. In a similar sense, the tetrameric nature of streptavidin makes it a bulky and non-optimal protein for use in a translational fusion protein. This is because bulkier fusion proteins tend to exhibit a higher risk of aggregation and instability, which is accentuated by the presence of multiple subunits. To mitigate these complications of working with streptavidin, __________ engineered a monomeric streptavidin using rational protein design. This engineered monomeric streptavidin (henceforth referred to as mSA2) eliminates the complications mentioned above, but displays a markedly lower affinity for biotin than its tetrameric counterpart (CITE). The existing part for monomeric streptavidin is BBa_K1896000.
This part is an improvement of BBa_K1896000. In hoping to further improve mSA2’s binding affinity to biotin for use in a diagnostic microfluidic assay, Waterloo iGEM (2021) used a computational workflow to perform rational protein design on mSA2. Each amino acid of mSA2 was iteratively mutated using Rosetta to determine the mutations yielding the lowest computational energy scores for mSA2, indicating the greatest stability. Only mutations occurring in the binding site were ultimately considered, as any other mutations were unlikely to affect binding affinity. Then, ligand docking was performed using AutoDock Vina to assess whether the mutations improved binding affinity between mSA2 and biotin; a lower complex energy corresponded to a higher binding affinity.
Ultimately, three binding site mutations were implemented: T74C, N12A, and Y52F. These mutations were able to decrease the energy of the mSA2-biotin complex by _______ kcal/mol compared to the unmutated mSA2. The following image depicts the improved mSA2, with the mutated residues highlighted:
IMAGE LINK
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]