Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.     

Development and characterization of a series of soluble tetrameric and monomeric streptavidin muteins with differential biotin binding affinities

The strong biotin-streptavidin interaction limits the application of streptavidin as a reversible affinity matrix for purification of biotinylated biomolecules. To address this concern, a series of single, double, and triple streptavidin muteins with different affinities to biotin were designed. The strategy involves mutating one to three strategically positioned residues (Ser-45, Thr-90, and Asp-128) that interact with biotin and other framework structure-maintaining residues of streptavidin. The muteins were produced in soluble forms via secretion from Bacillus subtilis. The impact of individual residues on the overall structure of streptavidin is reflected by the formation of monomeric streptavidin to different extents. Of the three targeted residues, Asp-128 has the most dramatic effect (Asp-128 > Thr-90 > Ser-45). Conversion of all three targeted residues to alanine results in a soluble biotin binding mutein that exists 100% in the monomeric state. Both wild-type and mutated (monomeric and tetrameric) streptavidin proteins were purified, and their kinetic parameters (on- and off-rates) were determined using a BIAcore biosensor with biotin-conjugated bovine serum albumin immobilized to the sensor chip. This series of muteins shows a wide spectrum of affinity toward biotin (K(d) from 10(-6) to 10(-11) m). Some of them have the potential to serve as reversible biotin binding agents.     

Design,production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.     

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop_3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (K_d) from 4×10⁻¹⁴ M to 4.45×10⁻¹⁰ M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.     

A monovalent streptavidin with a single femtomolar biotin binding site

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.     

Engineering a novel, stable dimeric streptavidin with lower isoelectric point

We have engineered a soluble, stable two-chain dimeric streptavidin (TCD) in Escherchia coli. Examination of the three-dimensional structure of streptavidin aided by empirical binding free-energy calculations helped us to select mutations at subunit interfaces that dissociate the native tetramer and stabilize the desired dimer. We chose positions W120, L124, V125 and H127 and mutated them to 120D/124D/125D/127D (TCD-1); 120D/124N/125S/127D (TCD-2); and 120D/124D/125S/127D (TCD-3). The H127D mutation creates electrostatic repulsion that disrupts the dimer-dimer interface, but leaves it very hydrophobic. Therefore, W120, L124 and V125 were mutated to hydrophilic residues to increase dimer solubility. Among the three candidates, TCD-2 gave the best result: a stable, active dimer with K(d) for biotin of approximately 1x10(-7)M after purification by gel-filtration chromatography. The experimental results confirm the possibility of rational engineering of low-pI dimeric streptavidins. Reduced-size streptavidin mutants with a net negative charge may be more suitable than antibodies or wild-type streptavidin for the targeting step in radioimmunotherapy because they should clear faster from the bloodstream and the kidney.     

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (k_off) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20–40% improvement in the measured k_off. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t_½) from 11 to 83 min at 20°C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

Engineered streptavidin monomer and dimer with improved stability and function

Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.     

Stable,high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K_d = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T_m = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.     

Reduced antibody response to streptavidin through site-directed mutagenesis

Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.     

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (K_d ~ 10^-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.     

Receptor Binding Activities of Biotinylated Derivatives of β-Nerve Growth Factor

beta-nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C-bio-NGF) using biotin hydrazide and the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution, using N-hydroxysuccinimidyl biotin, to produce derivatives with ratios of one, two, and four biotin moieties per NGF subunit (N-bio-NGF). The various biotinylated NGF derivatives were compared with native NGF for their capacity to compete with 125I-NGF for binding to NGF receptors on rat pheochromocytoma (PC12) cells at 4 degrees C. On the basis of such radioreceptor assays, C-bio-NGF was as effective as native NGF in binding to NGF receptors. C-bio-NGF was also as effective as native NGF in promoting neurite outgrowth from PC12 cells. In contrast, N-bio-NGF containing one biotin per NGF subunit was only 28% as active in binding as native NGF. Increasing the biotin:NGF ratio to 2 to 4 further decreased receptor binding to 13% and 6%, respectively, as compared to native NGF. Once bound to cells, C-bio-NGF had the capacity to mediate the specific binding of 125I-streptavidin to PC12 cells. This binding of streptavidin was prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. In addition, a variant PC12 line lacking functional NGF receptors was not labeled by 125I-streptavidin after prior incubation with C-bio-NGF.     

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.     

Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

Rational design of an active avidin monomer

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 --> 2 interface was mutated to lysine and asparagine 54 in the 1 --> 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K(d) approximately 10(-15) m of the wild-type tetramer to K(d) approximately 10(-7) m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.     

Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (Il2) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined. All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold-improved renaturation yield. One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and high-affinity receptor binding. The binding to the low-affinity Il2-binding protein (Tac antigen) was unimpaired. A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high- and the low-affinity receptor, however, was reduced about 20-fold. Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins. It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

Effects of tryptophan residue fluorination on streptavidin stability and biotin–streptavidin interactions via molecular dynamics simulations

Due to its highly specific and very strong binding, the (strept)avidin–biotin system forms the basis for numerous applications in the life sciences: immunoassays, DNA detection systems, affinity chromatography, etc. Fine-tuning of the ligand binding abilities of this system might provide new technologies with relevance to nanoscale research. Here, we report our computational investigations on wild type (WT) and modified streptavidin (SAV), assessing the impact of fluorination of tryptophan residues on biotin binding ability. Complexes of biotin with four SAV protein variants (WT-SAV, 4fW-SAV, 5fW-SAV and 6fW-SAV) were studied. We found that protein stability and folding are predicted to be weakly affected by fluorination. The host protein binding pocket decreases its ability to form numerous hydrogen bonds to biotin in the case of the 4fW-SAV variant. Conversely, the 5fW-SAV mutant is predicted to have an even more stable ligand–host hydrogen bonding network than WT-SAV. Thermodynamic perturbation investigations predict a decrease in biotin binding free energy from 3.0 to 6.5 kcal/mol per tetrameric host, with the 5fW-SAV mutant being least affected. Overall, the computational findings indicate that 6fW-SAV and, especially, 5fW-SAV to be promising variants of streptavidin for potential modifiable picomolar binding of the biotin ligand family. Figure Hydrogen bonding framework of the biotin–streptavidin system Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.