Difference between revisions of "Part:BBa K3843005:Design"

Revision as of 18:19, 18 October 2021

Affinity-improved monomeric streptavidin (mSA2+)

Design Notes

To obtain the affinity-improved sequence, the original protein was first computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Next, the protein's binding affinity to biotin was assessed using docking simulations in AutoDock Vina, which returned an energy score for the biotin+mSA2 complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of mSA2. The mutation and evaluation process was repeated until ultimately three mutations were implemented: T74C, N12A, and Y52F. The final protein was back-translated and codon optimized for E. coli K12. As well, illegal restriction sites for RFC[25] were removed to facilitate fusion protein construction.

Source

Improvement of binding affinity of monomeric streptavidin BBa_K1896000. Sequence obtained through computational rational protein design, as described above. The original sequence used as a basis for computational rational protein design was not the DNA sequence, but rather the amino acid sequence of monomeric streptavidin (PDB ID: 4JNJ, https://www.ncbi.nlm.nih.gov/Structure/pdb/4JNJ). After applying the desired mutations to 4JNJ, the mutated amino acid sequence was back-translated and codon-optimized.

References

DeMonte, D., Drake, E. J., Lim, K. H., Gulick, A. M., & Park, S. (2013, June 17). Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins, 81(9), 1621-33. https://onlinelibrary.wiley.com/doi/10.1002/prot.24320.