Part:BBa_K1896000

Monomeric Streptavidin (mSA2)

Codes for a monomeric variant of the tetrameric biotin-binding protein Streptavidin.

Usage and Biology

Glass slides coated with biotinylated PLA, dipped in crude cell lysate and then washed with saline. Left: the mGFPuv2-mSA2 fusion protein adheres to the PLA, right: the mGFPuv2 control easily washes off.

This part can be used to create fusion proteins that strongly bind to biotin and biotin-coated structures. The UGent Belgium 2016 iGEM team used this sequence in a GFP-mSA2 fusion protein that was shown to bind to biotin coated polylactic acid (PLA), a biodegradable polymer.

Streptavidin, originally isolated from Streptomyces avidinii, is used extensively in molecular biology due to its extraordinary affinity towards biotin (K_d ~10^-14M).[1] This interaction is used in vitro to purify various biomolecules to which biotin has been linked. When used in fusion proteins however, the tetrameric nature of Streptavidin can cause problems as the formed protein complexes can form aggregates that are toxic to the cell. This could be an explanation for the growth defects caused by BBa_K1896017 for example. For this reason, monomeric streptavidin variants have been developed using extensive protein engineering.

In the core streptavidin sequence, which can be seen in BBa_K283010, mutations were first introduced to replace short interfacial residues with longer charged residues in order to create electrostatic repulsion between the monomers. The resulting monomeric proteins however, tend to aggregate because of newly exposed hydrophobic residues, so mutations were introduced to replace these with polar residues. The monomers were further stabilised by introducing more charged residues that form salt bridges that stabilise the beta barrel structure.[2]

The resulting monomeric protein did however lose much of its affinity to biotin. To remedy this, researchers then created a hybrid sequence that substitutes key binding site residues with the sequences found in Rhizavidin, a naturally dimeric biotin binding protein from Rhizobium etli.[3] This exposed new hydrophobic residues, which were again replaced by polar amino acids. A final mutation near the binding site was found to increase the dissociation time of biotin and the resulting monomeric steptavidin, mSA2, was used in this part.[4]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal AgeI site found at 379
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., & Salemme, F. R. (1989). Structural origins of high-affinity biotin binding to streptavidin. Science, 243(4887), 85.
2. Lim, K. H., Huang, H., Pralle, A., & Park, S. (2011). Engineered streptavidin monomer and dimer with improved stability and function. Biochemistry, 50(40), 8682-8691.
3. Lim, K. H., Huang, H., Pralle, A., & Park, S. (2013). Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnology and bioengineering, 110(1), 57-67.
4. Mann, J. K., Demonte, D., Dundas, C. M., & Park, S. (2016). Cell labeling and proximity dependent biotinylation with engineered monomeric streptavidin. Technology, 1-7.

Waterloo iGEM 2021 improved the binding affinity of mSA2 to biotin using computational rational protein design methods. The following mutations were incorporated: ____, resulting in ______. The Part page for the improved mSA2 (dubbed mSA2+) is BBa_K3843005.