Engineering a disulfide-gated switch in streptavidin enables reversible binding without sacrificing binding affinity.

Although high affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is also required. To combine extremely tight and reversible binding, we have introduced disulfide bonds into opposite sides of a flexible loop critical for biotin binding, creating streptavidin muteins (M88 and M112) with novel disulfide-switchable binding properties. Crystal structures reveal how each disulfide exerts opposing effects on structure and function. Whereas the disulfide in M112 disrupts the closed conformation to increase koff, the disulfide in M88 stabilizes the closed conformation, decreasing koff 260-fold relative to streptavidin. The simple and efficient reduction of this disulfide increases koff 19,000-fold, thus creating a reversible redox-dependent switch with 70-fold faster dissociation kinetics than streptavidin. The facile control of disulfide formation in M88 will enable the development of many new applications requiring high affinity and reversible binding.

A bio-coupling approach using a dextran-binding domain to immobilize an engineered streptavidin to Sephadex for easy preparation of affinity matrix.

An engineered streptavidin, SAVSBPM18 with reversible biotin binding capability, has been successfully applied to purify biotinylated and streptavidin-binding peptide (SBP) tagged proteins. To simplify the preparation for the SAVSBPM18 affinity matrix without chemical conjugation, two bio-coupling approaches were developed based on a 14-kDa dextran-binding domain (DBD) from a Leuconostoc mesenteroides dextransucrase. The first approach offers simplicity for bio-coupling by creating a direct fusion, SAVSBPM18-Linker-DBD. Purification of the fusion from crude extract and its immobilization to Sephadex can be consolidated in one-step. The second approach aims at flexibility. A SnoopCatcher (SC) was fused to DBD to create SC-Linker-DBD. This fusion can covalently capture any recombinant proteins tagged with a SnoopTag (ST) including SAVSBPM18-Linker-ST via the formation of an isopeptide bond at the interface through the SnoopCatcher-SnoopTag interaction. Although monomeric DBD binds to dextran with nanomolar affinity, DBD tetramerized via streptavidin (SAVSBPM18-Linker-ST·SC-Linker-DBD) showed an even tighter binding to Sephadex. The majority of the fluorescently labelled DBD tetramers were retained on the Sephadex surface even after four months. Affinity columns generated using either approach effectively purified both SBP-tagged and biotinylated proteins. These columns are reusable and functional even after a year of frequent use.

A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix.

SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain. This domain was fused to the C-terminal end of SAVSBPM18. It allows the SAVSBPM18 fusions to bind reversibly to agarose and can serve as an affinity handle for purification of the fusion. Fluorescently labeled SAVSBPM18 fusions were found to be stably immobilized on Sepharose 6B-CL. The enhanced immobilization capability of the fusion to the agarose beads results from the avidity effect mediated by the tetrameric nature of SAVSBPM18. This approach allows the consolidation of purification and immobilization of SAVSBPM18 fusions to Sepharose 6B-CL in one step for affinity matrix preparation. The resulting affinity matrix has been successfully applied to purify both SBP tagged ß-lactamase and biotinylated proteins. No significant reduction in binding capacity of the column was observed for at least six months.

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 (Kd ~ 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.

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. Loop3-4 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 (Kd) 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.

The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits.

The 38-residue SBP-Tag binds to streptavidin more tightly (K(d) -/= 2.5-4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12-14) and a C-terminal HPQ sequence (residues 31-33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17-28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1-10 and 35-38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11-34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of ß-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag-streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

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.

Engineering monomeric streptavidin and its ligands with infinite affinity in binding but reversibility in interaction.

Natural tetrameric streptavidin captures and immobilizes biotinylated molecules with ultra-tight binding (K(d) approximately 10(-13) to 10(-14) M). In contrast, engineered monomeric streptavidin offers reversible binding (K(d) approximately 10(-7) M). To develop an ideal engineered streptavidin which possesses both the immobilization capability of the natural streptavidin and the reversible interaction reactivity of the monomeric streptavidin, a pair of engineered biomaterials was designed through molecular modeling. This system consists of two recombinant components: an engineered monomeric streptavidin M6, which has a cysteine residue (C118) near the biotin binding site, and a cysteine containing biotinylation tag. Interactions between M6 and the biotinylated peptide tag go through a two-stage process (capture and immobilization) to generate a covalently linked complex. Biotinylation is essential in the capture stage. Once the biotin moiety in the biotinylated tag is captured by M6, the biotinylated tag can fold back and rotate on the surface of the complex with the biotinylated lysine in the peptide tag as the axis until the formationof a disulfide bond. Consequently, cysteine residue in different positions flanking the biotin residue in the biotinylation tag can successfully form a disulfide bond with M6. Intermolecular disulfide bond formation between M6 and the tag containing protein offers the immobilization capability to M6. In the presence of reducing agent and biotin, bound ligands can be dissociated. This system has the potential to extend the biotin-streptavidin technology to develop reusable biosensor/protein chips and bioreactors.

Development of a LytE-based high-density surface display system in Bacillus subtilis.

The three N-terminal, tandemly arranged LysM motifs from a Bacillus subtilis cell wall hydrolase, LytE, formed a cell wall-binding module. This module, designated CWBM(LytE), was demonstrated to have tight cell wall-binding capability and could recognize two classes of cell wall binding sites with fivefold difference in affinity. The lower-affinity sites were approximately three times more abundant. Fusion proteins with ß-lactamase attached to either the N- or C-terminal end of CWBM(LytE) showed lower cell wall-binding affinity. The number of the wall-bound fusion proteins was less than that of CWBM(LytE). These effects were less dramatic with CWBM(LytE) at the N-terminal end of the fusion. Both CWBM(LytE) and ß-lactamase were essentially functional whether they were at the N- or C-terminal end of the fusion. In the optimal case, 1.2 × 10(7) molecules could be displayed per cell. As cells overproducing CWBM(LytE) and its fusions formed filamentous cells (with an average of nine individual cells per filamentous cell), 1.1 × 10(8)ß-lactamase molecules could be displayed per filamentous cell. Overproduced CWBM(LytE) and its fusions were distributed on the entire cell surface. Surface exposure and accessibility of these proteins were confirmed by immunofluorescence microscopy.

Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins.

Monomeric forms of avidin and streptavidin [(strept)avidin] have many potential applications. However, generation of monomeric (strept)avidin in sufficient quantity is a major limiting factor. We report the successful intracellular production of an improved version of monomeric streptavidin (M4) in a soluble and functional state at a level of approximately 70 mg/L of an Escherichia coli shake flask culture. It could be affinity purified in one step using biotin agarose with 70% recovery. BIAcore biosensor analysis using biotinylated bovine serum albumin confirmed its desirable kinetic properties. Two biotinylated proteins with different degrees of biotinylation (5.5 and 1 biotin per protein) pre-mixed with cellular extracts from Bacillus subtilis were used to examine the use of M4-agarose in affinity purification of protein. Both biotinylated proteins could be purified in high purity with 75-80% recovery. With the mild elution and matrix regeneration conditions, the M4-agarose had been reused four times without any detectable loss of binding capability. The relatively high-level overproduction and easy purification of M4, excellent kinetic properties with biotinylated proteins and mild procedure for protein purification make vital advancements in cost-effective preparation of monomeric streptavidin affinity matrix with desirable properties for purification of biotinylated molecules.

Engineering soluble monomeric streptavidin with reversible biotin binding capability.

Monomeric streptavidin with reversible biotin binding capability has many potential applications. Because a complete biotin binding site in each streptavidin subunit requires the contribution of tryptophan 120 from a neighboring subunit, monomerization of the natural tetrameric streptavidin can generate streptavidin with reduced biotin binding affinity. Three residues, valine 55, threonine 76, and valine 125, were changed to either arginine or threonine to create electrostatic repulsion and steric hindrance at the interfaces. The double mutation (T76R,V125R) was highly effective to monomerize streptavidin. Because interfacial hydrophobic residues are exposed to solvent once tetrameric streptavidin is converted to the monomeric state, a quadruple mutein (T76R,V125R,V55T,L109T) was developed. The first two mutations are for monomerization, whereas the last two mutations aim to improve hydrophilicity at the interface to minimize aggregation. Monomerization was confirmed by four different approaches including gel filtration, dynamic light scattering, sensitivity to proteinase K, and chemical cross-linking. The quadruple mutein remained in the monomeric state at a concentration greater than 2 mg/ml. Its kinetic parameters for interaction with biotin suggest excellent reversible biotin binding capability, which enables the mutein to be easily purified on the biotin-agarose matrix. Another mutein (D61A,W120K) was developed based on two mutations that have been shown to be effective in monomerizing avidin. This streptavidin mutein was oligomeric in nature. This illustrates the importance in selecting the appropriate residues and approaches for effective monomerization of streptavidin.

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro.

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.

A fast-acting, modular-structured staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy.

To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.

Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production.

To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.

Secretory production and purification of functional full-length streptavidin from Bacillus subtilis.

Streptavidin is a versatile molecule for many in vitro and in vivo applications. To optimize the production of the full-length streptavidin in a soluble and functional form via secretion using Bacillus subtilis as the expression host, three different strategies were used. These strategies include the construction of a synthetic streptavidin gene, the installation of a transcription terminator, and the use of a sporulation mutant strain. In comparison with the wild-type streptavidin gene in expression studies, a combination of these approaches resulted in a 2.3-fold increase in streptavidin production. The production yields in complex and semidefined media were 94 and 24 mg/liter, respectively. A simple purification scheme which requires only a single ion-exchange matrix was designed to purify streptavidin to homogeneity directly from the culture supernatant. Purified streptavidin was in full length with good biotin binding capacity (3.2 binding sites available per tetramer). A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional streptavidin for characterizations and practical applications.

Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion.

A major attraction in using Bacillus subtilis as an expression host for heterologous protein production is its ability to secrete extracellular proteins into the culture medium. To take full advantage of this system, an efficient method for recovering the target protein is crucial. For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification. The availability of large amounts of quality BirA can be critical for in vitro biotinylation. We report here the engineering and production of an Escherichia coli BirA and its application in the purification of staphylokinase, a fibrin-specific plasminogen activator, from the culture supernatant of Bacillus subtilis via in vitro biotinylation. BirA was tagged with both a chitin-binding domain and a hexahistidine tail to facilitate both its purification and its removal from the biotinylated sample. We show in this paper how, in a unique way, we solved the problem of protein aggregation in the E. coli BirA production system to achieve a yield of soluble functional BirA hitherto unreported in the literature. Application of this novel BirA to protein purification via in vitro biotinylation in general will also be discussed. Biotinylated staphylokinase produced in the study not only can act as an intermediate for easy purification, it can also serve as an important element in the creation of a blood clot targeting and dissolving agent.

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.