Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: applications in flow cytometry and ELISA.

Monomeric streptavidin (mSA) offers a combination of structural and binding properties that are useful in many applications, including a small size and monovalent biotin binding. Because mSA contains a structurally important disulfide bond, the molecule does not fold correctly when expressed inside the cell. We show that mSA can be expressed in a functional form in Escherichia coli by fusing the OmpA signal sequence at the amino terminus. Expressed mSA is exported to the periplasm, from which the molecule leaks to the medium under vigorous shaking. Purified mSA can be conjugated with FITC and used to label microbeads and yeast cells for analysis by flow cytometry, further expanding the scope of mSA-based applications. Some applications require recombinant fusion of mSA with another protein. mSA fused to EGFP cannot be secreted to the medium but was successfully expressed in an engineered cell line that supports oxidative folding in the cytoplasm. Purified mSA-EGFP and mSA-mCherry bound biotin with high affinity and were successfully used in conventional flow cytometry and imaging flow cytometry. Finally, we demonstrate the use of mSA in ELISA, in which horseradish peroxidase-conjugated mSA and biotinylated secondary antibody are used together to detect primary antibody captured on an ELISA plate. Engineering mSA to introduce additional lysine residues can increase the reporter signal above that of wild-type streptavidin. Together, these examples establish mSA as a convenient reagent with a potentially unique role in biotechnology.

RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.

Synthetic biology and metabolic engineering seek to re-engineer microbes into "living foundries" for the production of high value chemicals. Through a "design-build-test" cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

Biotin-independent strains of Escherichia coli for enhanced streptavidin production.

Biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. However, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. Moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. Here we describe the creation of biotin-independent strains of Escherichia coli and Corynebacterium glutamicum through installation of an optimized malonyl-CoA bypass, which re-routes natural fatty acid synthesis, rendering the previously essential vitamin completely obsolete. We utilize biotin-independent E. coli for the production of the high-value protein streptavidin which was hitherto restricted because of toxic effects due to biotin depletion. The engineered strain revealed significantly improved streptavidin production resulting in the highest titers and productivities reported for this protein to date.

GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris.

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol-based processes lead to large proportions of biotin-blocked binding sites of streptavidin due to biotin-supplemented media. Targeting these problems, this paper provides strategies for the methanol-free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin-blocked streptavidin. The optimized, easily scalable fed-batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin-free streptavidin and a productivity of 57.8 nM h(-1) based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un-optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (µ ≈ D < 0.01 h(-1) ), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855-864, 2016.

Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli 'leaky mutant'.

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic 'leaky mutant' E. coli JW1667-5 (Δlpp-752:kan) at 30°C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton(®) X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790±166nM were reached in shake flasks at a productivity of 49.6nMh(-1). Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608±169nM and a productivity of 65.2nMh(-1) in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3±4.3% indicated a highly bioactive product.

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.

Affinity purification strategies for proteomic analysis of transcription factor complexes.

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein-protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

Killed Bacillus subtilis spores expressing streptavidin: a novel carrier of drugs to target cancer cells.

Carriers of drugs in cancer therapy are required to reduce side-effects of the drugs to normal cells. Here we constructed killed recombinant Bacillus subtilis spores (SA1) that expressed streptavidin as a chimeric fusion to the spore coat protein CotB and used the spores as bioparticle carrier. When bound with biotinylated cetuximab these spores could specifically target to the epidermal growth factor receptor on HT 29 colon cancer cells, thereby delivered paclitaxel to the cells with 4-fold higher efficiency, as indicated by fluorescent intensity of paclitaxel Oregon Green 488 bound to HT29 cells. Based on real-time monitoring of cell index, the IC50 of growth of HT29 cells by paclitaxel-SA1-cetuximab was estimated to be 2.9 nM approximately 5-fold lower than water-soluble paclitaxel (14.5 nM). Instability of DNA content was observed when cells were treated with 16 nM paclitaxel-SA1-cetuximab, resulting in a 2-fold enhancement in polyploidy cells. Thus, by targeting the release of paclitaxel to HT29 cells, spore-associated cetuximab augmented the inhibitory effect of paclitaxel on cell division and proliferation. The SA1 could be used as a "universal" drug carrier to target specific biomarkers on cancer cells by conjugating with suitable biotinylated antibodies.

Preparation and functional evaluation of RGD-modified streptavidin targeting to integrin-expressing melanoma cells.

The vertical growth stage is the most dangerous stage of melanoma and is often associated with a poor prognosis. The increased invasiveness and metastasis that is typical for vertically growing melanoma are mediated by the molecules of cell adhesion (particularly, integrins). Integrin αvß3, which is abundantly expressed on melanoma cells with high metastatic potentials and is characterized by low expression levels in normal melanocytes, is potentially an attractive target for melanoma diagnostics and therapy. Integrin αvß3 is known to recognize the arginine-glycine-aspartic (RGD) sequence, which has been found in a wide variety of its natural ligands. Here expression vectors bearing the genes of fusion proteins have been constructed for producing these proteins in Escherichia coli. Such fusion proteins consist of a peptidic 'address,' targeting the integrins on melanoma cells, linked to an 'adaptor' for the attachment of a diagnostic or toxic agent. The peptidic 'address' contains the RGD motif, which is stabilized by a disulfide bond to achieve the optimal receptor binding conformation. The 'adaptor' is a tetrameric protein, namely, streptavidin, that is able to achieve high-affinity binding of d-biotin (K(d) = 10(-15) M) and confer avidity to the address peptide. This binding ability facilitates the generation of anti-melanoma diagnostic and therapeutic agents using the appropriate biotin derivatives. These recombinant proteins were purified from the periplasm of E.coli using columns with 2-iminobiotin agarose and demonstrated an ability to adhere to the surface of murine and human melanoma cells.

Development of fed-batch strategies for the production of streptavidin by Streptomyces avidinii based on power input and oxygen supply studies.

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains. However, little is known about the influence of power input and oxygen supply as well as feeding strategies on the production of streptavidin by S. avidinii. This paper provides a systematic analysis of the effect of rotary frequency of the stirrer, leading to a plateau-like streptavidin formation behaviour between 400 and 700 min(-1). This plateau was characterized by specific power inputs between 79 and 107 W L(-1) and corresponding maximal product concentrations of 6.90 µM in 6 days. Lower as well as higher rotary frequencies were not beneficial. Subsequently, a linear fed-batch procedure could be established reproducibly yielding 39.20 µM streptavidin in 14 days, characterized by a constant productivity of 114 nM h(-1). Fed-batch procedures based on dissolved oxygen were less efficient. The linear feeding strategy presented in this paper led to the highest streptavidin concentration ever reported and exceeded the maximal product level given in the literature drastically by a factor of 8.5.

Cloning and expression of a functional core streptavidin in Pichia pastoris: strategies to increase yield.

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min(-1) , and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L(-1) ). These parameters yielded 4.0 g L(-1) of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L(-1) of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.

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.

[Expression, purification and bioactivity evaluation of streptavidin-tagged human interleukin-21 fusion protein].

OBJECTIVE: To obtain streptavidin-tagged human interleukin-21 (hIL21) fusion protein and evaluate its bioactivities. METHODS: hIL21-SA-pET21 and pET24a-SA- hIL21 plasmids were constructed and expressed in BL21(DE3) host bacteria. The hIL21-SA and SA- hIL21 fusion protein were purified through Ni-NTA affinity chromatography and refolded by dialysis. Flow cytometry was used to detect hIL21-SA and SA- hIL21 fusion protein on the biotinylated MB49 tumor cells. MTT assay was used to evaluate the effect of the fusion protein on the proliferation of human peripheral blood lymphocytes (PBLs) stimulated by Anti-CD3. RESULTS: The recombinant fusion proteins were highly expressed in BL21(DE3) at about 30% of the total bacterial proteins. The two fusion proteins exhibited bifunctional activities, i.e. both biotin-binding property and hIL21 activity and SA-mediated high-affinity binding to biotinylated cell surfaces (with anchoring modified rate of about 95.18% and 96.91%). CONCLUSION: We have successfully obtained bifunctional fusion protein hIL21-SA and SA- hIL21,which will provide a basis for further study of tumor biotherapy using the proteins.

[Preparation and bioactivity evaluation of streptavidin-tagged human interleukin-15 fusion protein].

OBJECTIVE: To obtain streptavidin-tagged human interleukin-15 (SA/hIL15) fusion protein and evaluate its bioactivity. METHODS: pET24a-6His-SA-hIL-15 and pET32a-hIL-15-SA-6His plasmids were constructed and expressed in BL 21(DE3) host bacteria to generate the fusion protein. The recombinant fusion protein IL-15/SA was purified using Ni-NTA affinity chromatography and refolded, and the efficiency of surface modification of the fusion protein on biotinylated cells was examined by fluorescence-activated cell sorting. CCK-8 method was used to evaluate the effect of IL-15/SA fusion protein in inducing the proliferation of human peripheral-blood lymphocyte (PBL) cells stimulated by PHA. RESULTS: The recombinant SA-hIL-15 and hIL15-SA fusion proteins were highly expressed in BL21(DE3) at about 20% of the total bacterial proteins. The purified hIL15-SA fusion protein exhibited a bifunctionality by promoting the proliferation of PBL cells activated by PHA and high-affinity binding to biotinylated cell surface mediated by SA, with a cell surface modification efficiency exceeding 95%. SA-hIL-15 showed a 4-fold higher hIL15 bioactivity than hIL15-SA. CONCLUSION: SA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surface-modified cancer cell vaccine.

[Expression, purification and refolding of streptavidin-tagged human tumor necrosis factor-alpha fusion protein].

OBJECTIVE: To study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein. METHODS: SA-TNF-alpha fusion protein was expressed in BL21(DE3) host bacteria, purified using Ni-NTA affinity chromatography and refolded by dilution and dialysis followed by identification using Western blotting. The effect of SA-TNF-alpha fusion protein against L929 cells was evaluated by MTT assay. Flow cytometry was used to analyze the surface modification of biotinylated MB49 tumor cells by SA-TNF-alpha fusion protein. RESULTS: Recombinant SA- TNF-alpha fusion protein was expressed in BL21(DE3) at about 30% of total bacterial protein, with a purity of about 95% after purification. The SA-TNF-alpha fusion protein existed as dimmers, tetramers and higher order structures after refolding. The fusion protein exhibited a bi-functionality by inhibiting L929 cells and SA-mediated high-affinity binding to biotinylated cell surfaces, with an anchor modification rate of above 90%. CONCLUSION: The dimmers, tetramers and higher order structures of the obtained SA-TNF-alpha fusion protein all exhibit a bi-functionality, and may serve as a potential candidate therapeutic agent for tumors.

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.

Brave new (strept)avidins in biotechnology.

Avidin and streptavidin are widely used in (strept)avidin-biotin technology, which is based on their tight biotin-binding capability. These techniques are exceptionally diverse, ranging from simple purification and labeling methods to sophisticated drug pre-targeting and nanostructure-building approaches. Improvements in protein engineering have provided new possibilities to develop tailored protein tools. The (strept)avidin scaffold has been engineered to extend the existing range of applications and to develop new ones. Modifications to (strept)avidins--such as simple amino acid substitutions to reduce biotin binding and alter physico-chemical characters--have recently developed into more sophisticated changes, including chimeric (strept)avidins, topology rearrangements and stitching of non-natural amino acids into the active sites. In this review, we highlight the current status in genetically engineered (strept)avidins and illustrate their versatility as advanced tools in the multiple fields of modern bioscience, medicine 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.

A robust method for production of MHC tetramers with small molecule fluorophores.

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.

Efficient incorporation of a nonnatural amino acid into a protein in an insect cell-free translation system.

Recently, we have succeeded in incorporating various nonnatural amino acids into proteins by using four-base codon-anticodon pairs in Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system. Here, the reaction was conducted under various conditions in order to optimize the incorporation efficiency. The optimal concentration of aminoacyl-tRNA, reaction temperature, and reaction time were 2 nM, 25 degrees C, and 1.5 hr, respectively.