Team:LMU-TUM Munich/Proteins

Interaction of biotin and Streptavidin leads to a pseudopolymerization

Muc16 ProteinFigure 002.png


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Protein produced and characterized

Muc16 ProteinOverview 001.png


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Streptavidin and Biotin: The strongest non covalent binding in nature

Streptavidin is a tetrameric protein with a molecular weight of 52.8 kDa[1], it can be isolated from Streptomyces avidinii. Each subunit is able to bind one molecule of biotin (molecular weight = 244.3 Da). This specific, non-covalent bondage with a femto molar dissociation constant (kD = 10-15 M) is one of the strongest known biological affinities. Antibodies, in comparison, have lower dissociation constants with 10-7 – 10-11 M.
One subunit of streptavidin is organized as an eight stranded antiparallel beta sheet of coiled polypeptide chains which form a hydrogen bonded barrel with extended hydrogen loops[2]. The biotin binding pocket consists of primarily aromatic or polar side chains, these interact with the hetero atoms of biotin.
Due to its low unspecific binding, Streptavidin is primarily used in protein-purification and biochemical analytic.[3] Biotinylation does not interrupt functions of a biomolecule, the small molecule can be attached to lysine residues chemically or enzymatic (Biotin ligase).
The rapid and irreversible linkage is generally due to multiple hydrogen bonds as well as van der Waals interactions. Comparing the structures of apostreptavidin and the streptavidin-biotin complex, determined by multiple isomorphous replacement, provides several structural differences of the binding pocket. The ordering of two flexible surface polypeptide loops results in burying the biotin molecule in its pocket. Without biotin the L3/4 loop is disordered and does not give clear electron density, it closes upon biotin binding.[4].
Important for the high barrier of dissociation are biotin-tryptophan contacts in the binding pocket. There are four significant tryptophan residues involved in the binding site. The three residues Trp79, Trp92 and Trp108 are lined up in one section. Site directed mutagenesis states that W79F and W108F show small ΔΔG°. Trp120, which binds the biotin molecule from an adjacent subunit, displays a much larger influence in binding free energy[5].
Moreover the hydrogen bonding network and van der waals interactions show great influence in binding free energy. Compared to similar hydrogen bonding donors and acceptors of other protein-ligand systems the hydrogen bonding of Streptavidin to the ureido oxygen of biotin is remarkably high.

Application in our project

We use the extraordinary strength of the biotin:streptavidin binding for linking genetically engineered cells with streptavidin. The cells express a receptor which allows them to present a biotin molecule or a streptavidin variant on their cell surface (link receptor construct). The tetrameric Protein itself is able to cross link the biotinylated cells and form a stable network. A linker molecule like biotinylated PAS-Lysin, however, might increase the stability of the cell structure (link PAS)

Streptavidin variantes and homologes

protein residues molecular weight monomer [Da] molecular weight tetramer [Da] theoretical pI pI (IEF) extinction coefficient (monomer, A280) [M-1 cm-1]
Streptavidin wt 126 13200.34 52801.36 6.09 41940
Traptavidin 126 13129.22 52516.88 5.14 41940
Strep-Tactin 126 13241.44 52516.88 8.32 41940
enhanced monomeric Avidin 138 15220.30 / 5.91 35075
single chain Avidin 567 61530.65 / 5.91 94460

Traptavidin

is a streptavidin mutant (S52G, R53D) and shows a 10 times lower dissociation constant (KD= 10-16 M) and increased mechanical strength of the biotin binding. Moreover, it has improved thermostability before splitting into monomers (~10 °C higher)[6]. In contrast to streptavidin, the Traptavidin L3/4 loop (residues 45-50) does not change its conformation while binding biotin. The binding pocket is already closed and lacks flexibility. The loss of a structural change may decrease the entropic cost of binding and inhibits dissociation. The traptavidin:biotin dissociation at pH 7.4 is significantly slower than streptavidin:biotin dissociation. The hydrogen bonding length of both proteins to biotin are comparable.

Figure 5: dissociation of Avidin, Streptavidin and Traptavidin

Strep-Tactin

is an engineered streptavidin variant, which can bind a specific peptide sequence, called Strep tag. The Peptide sequence is eight residues long and can be fused N- or C-terminal to recombinant protein by subcloning. Using the Strep tag-Strep-Tactin-System for affinity chromatography provides great yields in protein- or protein complex purification. The strep-tagged protein binds Strep-Tactin, which is immobilized on the column. After washing, the protein can be eluted with biotin, which binds the Strep-Tactin several orders of magnitude stronger. The Strep tag (amino acid sequence: WRHPQFGG) was primarily designed to bind Streptavidin, over the years both the Strep tag and the Streptavidin were optimized (strep tag II: WSHPQFEK) [7]

Enhanced Monomeric Avidin (eMA)

was engineered for applications like molecular labelling without unwanted cross-linking of biotin conjugats. The monomeric protein, however, does not consist of a monomeric streptavidin subunit. Streptavidin binding pockets depend on one residue from a adjacent subunit, thus binding affinity of a single subunit is decreased (KD = 10-7 to 10-9 M). eMA consists of a monomerized Rhizavidin dimer, which contains a disulfid bond in the binding site restraining the protein and forming a rigid binding site[8].

Single chain avidin (scAvidin)

Production and purification of streptavidin

The minimal Streptavidin wildtype gene is cloned into pASK111 plasmid, which is transcriptionally initiated by a T7-promoter. The E.coli BL-R (DE3) strain codes the T7-polymerase of a Phagosome, which can be induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG).
A shaking flask with 2 L LB medium is inoculated 1:40 with a 50 ml preculture of E.coli BL-R. The bacterial suspension shakes at 37 °C for a few hours until its optical density reaches the range between 0.5 and 1. At this point, there are enough cells for inducing protein production. After 4 hours or production over night the cells can be harvested by centrifugation (5000 rpm for 20 min).
The cell pellet is resuspended in a 20 mM Tris/HCl buffer (500 mM NaCl, pH 8). Homogenization can be proceeded with French press or PANDA.The cell lysate is centrifuged at 11500 rpm for 30 min to separate the inclusion bodies in the pellet from the soluble proteins of the supernatant.
The inclusion bodies consist of aggregated and misfolded protein, which are denaturated with 6 M guanidinium chloride and centrifuged at 15000 rpm for 15 minutes. The aggregations in the pellet are discarded and the supernatant is added slowly into a high volume of PBS (30 ml PBS for 1 ml protein solution) and incubated over night. Guanidinium chloride is diluted and proteins are able to fold correctly.The solution is centrifuged again (11500 rpm for 30 min) to remove aggregates.
The Streptavidin purification is proceeded by fractionally ammonium sulfate precipitation. After each precipitation step the solution is incubated without stirring for few hours and centrifuged at 11500 rpm for 30 minutes afterwards.
The first precipitation step is 40 % saturation (1.75 M). Streptavidin remains soluble, the pellet can be discarded. After centrifugation the ammonium sulfate saturation is increased up to 70 % (3.37 M), Streptavidin is no longer soluble and precipitates.
The pellet is resuspended in a 50 % saturated ammonium sulfate solution (2.25 M), streptavidin again precipitates. After centrifugation the pellet is resuspended in 1x PBS buffer. As a result the solution contains only those proteins, which precipitate between 40 % and 50 % ammonium sulfate saturation.
One last centrifugation at 15000 rpm for 30 minutes removes all insoluble impurities. The solution is dialysed against 20 mM Tris/HCl buffer without salt (pH 8). Streptavidin has an isoelectric point of 6.09, therefore it is negatively charged in this buffer and a positively charged ResQ column is used for ion exchange chromatography. An increasing salt concentration of the buffer causes fractional elution of protein.
Streptavidin monomers, tetramers and aggregates can be identified by gel filtration with a Sephacryl column. After sterile filtration, a UV/VIS spectrum of the solution is measured in order to determine its concentration.[9]
The production of Traptavidin, Strep-Tactin and enhanced monomeric Avidin is analogue.

RESULTS

  1. ↑ http://www.expasy.org/
  2. ↑ 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.
  3. ↑ Skerra, A., Gebauer, M., Schönfeld, D., Schmelz, E. (2007). Skript zu "Fortgeschrittenenpraktikum Proteinchemie"
  4. ↑ 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.
  5. ↑ Stayton, P. S., Freitag, S., Klumb, L. A., Chilkoti, A., Chu, V., Penzotti, J. E., ... & Stenkamp, R. E. (1999). Streptavidin–biotin binding energetics. Biomolecular engineering, 16(1), 39-44.
  6. ↑ 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.
  7. ↑ Schmidt, T. G., & Skerra, A. (2007). The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nature protocols, 2(6), 1528-1535.
  8. ↑ Lee, J. M., Kim, J. A., Yen, T. C., Lee, I. H., Ahn, B., Lee, Y., ... & Jung, Y. (2016). A Rhizavidin Monomer with Nearly Multimeric Avidin‐Like Binding Stability Against Biotin Conjugates. Angewandte Chemie International Edition, 55(10), 3393-3397.
  9. ↑ Skerra, A., Gebauer, M., Schönfeld, D., Schmelz, E. (2007). Skript zu "Fortgeschrittenenpraktikum Proteinchemie"