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.     

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.     

Expression and purification of soluble monomeric streptavidin in Escherichia coli

We recently reported the engineering of monomeric streptavidin (mSA) for use in monomeric detection of biotinylated ligands. Although mSA can be expressed functionally on the surface of mammalian cells and yeast, the molecule does not fold correctly when expressed in Escherichia coli. Refolding from inclusion bodies is cumbersome and yields a limited amount of purified protein. Improving the final yield should facilitate its use in biotechnology. We tested the expression and purification of mSA fused to GST, MBP, thioredoxin, and sumo tags to simplify its purification and improve the yield. The fusion proteins can be expressed solubly in E. coli and increase the yield by more than 20-fold. Unmodified mSA can be obtained by proteolytically removing the fusion tag. Purified mSA can be immobilized on a solid matrix to purify biotinylated ligands. Together, expressing mSA as a fusion with a solubilization tag vastly simplifies its preparation and increases its usability in biotechnology.     

抗CEA单链抗体与链亲和素融合基因的表达

克隆分泌CEA杂交瘤细胞重链可变区（V_H）和轻链可变区（V_L）,以Linker连接V_H及V_L构建抗CEA单链抗体.同时以Spacer连接单链抗体和链亲和素,构建成功单链抗体和链亲和素融合基因,克隆该融合基因至原核表达载体,pET21a(+),经IPTG诱导表达出该双特异性融合蛋白.活性鉴定表明该融合蛋白具有结合CEA及生物素的双特异性.该融合蛋白在生物领域中有较广阔的应用前景.     

Design and Characterization of a Membrane Protein Unfolding Platform in Lipid Bilayers

Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.     

Biotin-assisted folding of streptavidin on the yeast surface

Yeast surface display allows heterologously expressed proteins to be targeted to the exterior of the cell wall and thus has a potential as a biotechnology platform. In this study, we report the successful display of functional streptavidin on the yeast surface. Streptavidin binds the small molecule biotin with high affinity (K(d) ≈ 10(-14)M) and is used widely in applications that require stable noncovalent interaction, including immobilization of biotinylated compounds on a solid surface. As such, engineering functional streptavidin on the yeast surface may find novel uses in future biotechnology applications. Although the molecule does not require any post-translational modification, streptavidin is difficult to fold in bacteria. We show that Saccharomyces cerevisiae can fold the protein correctly if induced at 20°C. Contrary to a previous report, coexpression of anchored and soluble streptavidin subunits is not necessary, as expressing the anchored subunit alone is sufficient to form a functional complex. For unstable monomer mutants, however, addition of free biotin during protein induction is necessary to display a functional molecule, suggesting that biotin helps the monomer fold. To show that surface displayed streptavidin can be used to immobilize other biomolecules, we used it to capture biotinylated antibody, which is then used to immunoprecipitate a protein target.     

Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

Signal amplification in flow cytometry using biotin tyramine.

BACKGROUND: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents--normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to generate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. METHODS: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti-human CD4 and anti-human CD36 antibodies binding to either human lymphocytes or mixed mononuclear cells. RESULTS: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification. CONCLUSIONS: The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.     

Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.     

Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.     

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.     

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.     

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.     

Constrained cell recognition peptides engineered into streptavidin.

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.     

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.     

Development and effects of immunoliposomes carrying an antisense oligonucleotide against DHFR RNA and directed toward human breast cancer cells overexpressing HER2

The development and the effect of immunoliposomes directed against human breast cancer cells overexpressing p185/HER2 are described. These immunoliposomes carry an antisense oligonucleotide directed toward the translational start site of dihydrofalate reductase (DHFR) RNA, which causes high cytotoxicity. To prepare the immunoliposomes, we followed two methodologies based on the high affinity between streptavidin and biotin and the use of biotinylated antibodies. In the first approach, the streptavidin molecule is covalently attached to the phospholipid DOPE, which is mixed with the cationic liposome DOTAP complexed with the antisense oligonucleotide. The second approach, which is much easier to perform, involves the binding of streptavidin to antibody and oligonucleotide, both biotinylated, and the latter complexed with DOTAP. The formation of the intermediary complexes of this immunoliposome was studied sequentially by gel electrophoresis. The uptake of the oligonucleotide carried by the immunoliposome was monitored by flow cytometry and confocal microscopy. As a model, we used SKBR3 cells that overexpress p185. The full immunoliposomes were more toxic than the antisense oligonucleotide in the absence of the antibody, thus increasing the sensitivity of the treatment.     

Improvement of production,assay and purification of streptavidin

Summary The production of streptavidin byStreptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.     

Genetic engineering of streptavidin,a versatile affinity tag

Streptavidin, a tetrameric protein produced by Streptomyces avidinii, has been used as a useful, versatile affinity tag in a variety of biological applications. The efficacy of streptavidin is derived from its extremely high binding affinity for the vitamin biotin. For the last several years, we have used genetic engineering as a primary means to enhance the properties of streptavidin and to expand the application of streptavidin as an affinity tag. In this review, we describe several genetically engineered streptavidin variants, which include a streptavidin with a reduced biotin-binding affinity, a dimeric streptavidin, and a fusion protein between streptavidin and protein A, along with their potential applications in biological science.     

Development of new biotin/streptavidin reagents for pretargeting

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.