A whole area scanning-enabled direct-counting strategy for studying blocking efficiency in mitigating protein-solid surface binding.

The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.

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.

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-ß-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016.

Desarrollo de un ensayo sandwich de doble antígeno para la detección de anticuerpos contra el VIH-2 empleando un péptido sintético biotinilado de la proteína gp36 / Development of an antigen ‘sandwich’ enzyme immunoassay for the detection of antibodies against HIV-2 by using a biotinylated synthetic peptide of gp36 protein

INTRODUCCIÓN: Existen varios métodos para la detección de anticuerpos contra el virus de inmunodeficiencia humana (VIH), y entre estos se encuentra el ELISA tipo sandwich de doble antígeno, muy utilizado en la actualidad. El objetivo de este trabajo es evaluar un péptido sintético monomérico biotinilado de la glucoproteína de transmembrana gp36 del VIH-2, en un ensayo de sandwich, para la detección de anticuerpos contra la esta proteína del VIH-2. MATERIALES Y MÉTODOS: Para desarrollar el ensayo se utilizaron placas recubiertas con la proteína recombinante gp36 a 0,5μg/ml y con el péptido sintético gp36(5) a 1μg/ml; la concentración del péptido sintético gp36(5) biotinilado (gp36(5)-B) utilizada fue 0,1μg/ml, preparada con una solución regula- dora Tris-BSA-NaCl y el conjugado Estreptavidina-Fosfatasa Alcalina diluido 1:30.000 preparado con la solución PBS-Sacarosa-BSA. Se evaluaron muestras de suero positivas a anticuerpos contra los virus VIH- 1 y VIH-2 (88 y 34, respectivamente), 483 muestras negativas procedentes de donantes de sangre y 96 muestras de suero para evaluar la especificidad analítica. Todas las muestras fueron evaluadas en el UMELISA HIV1+2 RECOMBINANT, las que resultaron reactivas se confirmaron por el ensayo confirmatorio DAVIH-BLOT. RESULTADOS: Las 34 muestras con anticuerpos contra el VIH-2 fueron evaluadas como positivas en ambas variantes de recubrimiento; la mayor especificidad se obtuvo con la variante que empleó el péptido sintético gp36(5) en el recubrimiento. El ensayo sandwich de doble antígeno desarrollado empleando el gp36 (5)-B permite la detección de anticuerpos contra la proteína gp36 del VIH-2

INTRODUCTION: Among the several existing methods for the detection of antibodies to HIV, the 'sandwich' ELISA is currently the most used. This study aims to assess a biotinylated monomeric synthetic peptide of the glycoprotein transmembrane gp36 from HIV-2, in a sandwich assay, for the detection of antibodies against this HIV-2 protein. MATERIALS AND METHODS: To perform the assay, plates coated with recombinant protein gp36 at 0.5 μg/mL and synthetic peptide gp36(5) at 1 μg/mL were used. The concentration of the biotinylated synthetic pep- tide (gp36(5)-B) used was 0.1 μg/mL prepared with a Tris-BSA-NaCl buffer solution and the Streptavidin- Alkaline Phosphatase conjugate diluted 1:30000 prepared with a PBS-Sucrose-BSA solution. Positive serum samples to antibodies against HIV-1 and HIV-2 viruses (88 and 34, respectively) were tested, with 483 negative samples from blood donors and 96 serum samples to assess the analytical specificity. All the samples were tested using the UMELISA HIV 1+2 RECOMBINANT assay, and all positives were confirmed using a DAHIV-BLOT assay. RESULTS: Thirty four samples with antibodies against HIV-2 were assessed as positive for both coating variants. The highest specificity was obtained with the variant using the synthetic peptide gp36(5) in its coating. The antigen 'sandwich' assay developed by using gp36(5)-B enables the detection of antibodies against gp36 protein of HIV-2

A novel approach to make homogeneous protease-stable monovalent streptavidin.

The interaction between the tetramer streptavidin and biotin is recognized as one of the strongest non-covalent associations. Owing to the tight and specific binding, the streptavidin-biotin system has been used widely for bimolecular labeling, purification, immobilization, and even for targeted delivery of therapeutics drugs. Here, we report a novel approach to make homogeneous monovalent tetramer streptavidin. The purified monovalent protein showed both thermal stability and protease stability. Unexpectedly, we found that two proteases, Proteinase K (PK) and Subtilisin (SU), can efficiently remove the His8-tag from the wild-type subunit without affecting the tetramer architecture of monovalent streptavidin, thus making it more homogeneous. In addition, crystallization was performed to assure the homogeneity of the monovalent protein prepared. Overall, monovalent streptavidin shows increased homogeneity and will likely be valuable for many future applications in a wide range of research areas.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions.

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.

Ultrafast molecular motor driven nanoseparation and biosensing.

Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10 µm², with a concentration half-time of approximately 40 s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.

Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.

Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 Å resolution of Strep-Tactin in complex with desthiobiotin.

Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system.

The importance of viral and tumour vaccines in eliciting elicit strong CD8+ T-cell responses has been widely acknowledged. Strategies exploring ways to enhance CD8+ T-cell responses have been developed, including targeting of vaccine antigens to dendritic cell (DC) receptors to access to the cross presentation pathway. Many DC endocytic receptors could potentially lead to augmented CD8+ T-cell responses if antigens were targeted directly to them, however only a few receptors have been explored because current targeting reagents are limited in the number of receptors that they are able to target. Consequently, this study describes the production and purification of a streptavidin-fusion protein that provides a versatile and efficient means to target antigen to more than one DC receptor. A model antigen gene, CMV pp65, and a streptavidin core gene, were spliced together using an overlap-extension PCR technique. The resulting fusion gene was cloned into a vector allowing expression in an Adenovirus-based expression system. Expression was verified and optimised before Ni-NTA affinity chromatography purification. Evaluation of pp65-streptavidin immunogenicity revealed that it elicits similar levels of CD8+ T-cell proliferative responses as pp65 and is able to effectively target specific DC receptors when used in addition to biotinylated receptor-specific antibodies. Additionally, enhancement of CD8+ T-cell responses was shown after directing pp65-strep to selected DC receptors in preliminary in vitro experiments. Collectively, this highlights the ease of production of a streptavidin-fusion protein, and demonstrates its use as a promising strategy to evaluate numerous DC receptors as potential targets in vaccine strategies.

Nanoscale molecular traps and dams for ultrafast protein enrichment in high-conductivity buffers.

We report a new approach, molecular dam, to enhance mass transport for protein enrichment in nanofluidic channels by nanoscale electrodeless dielectrophoresis under physiological buffer conditions. Dielectric nanoconstrictions down to 30 nm embedded in nanofluidic devices serve as field-focusing lenses capable of magnifying the applied field to 10(5)-fold when combined with a micro- to nanofluidic step interface. With this strong field and the associated field gradient at the nanoconstrictions, proteins are enriched by the molecular damming effect faster than the trapping effect, to >10(5)-fold in 20 s, orders of magnitude faster than most reported methods. Our study opens further possibilities of using nanoscale molecular dams in miniaturized sensing platforms for rapid and sensitive protein analysis and biomarker discovery, with potential applications in precipitation studies and protein crystallization and possible extensions to small-molecules enrichment or screening.

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.

Design of a reversible biotin analog and applications in protein labeling, detection, and isolation.

To expand the applicability of the biotin-(strept)avidin system, a biotin analog with reversible binding under non-denaturing conditions has been designed, and its applications in protein labeling, detection, and isolation have been evaluated.

Supported bilayer electrophoresis under controlled buffer conditions.

A pH controlled flow cell device was constructed to allow electrophoretic movement of charged lipids and membrane associated proteins in supported phospholipid bilayers. The device isolated electrolysis products near the electrodes from the electrophoresis process within the bilayer. This allowed the pH over the bilayer region to remain within ±0.2 pH units or better over many hours at salt concentrations up to 10 mM. Using this setup, it was found that the electrophoretic mobility of a dye conjugated lipid (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE)) was essentially constant between pH 3.3 and 9.3. In contrast, streptavidin, which was bound to biotinylated lipids, shifted from migrating cathodically at acidic pH values to migrating anodically under basic conditions. This shift was due to the modulation of the net charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of a polyethylene glycol (PEG) cushion beneath the bilayer or the increase in the ionic strength of the buffer solution resulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE. As such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently of one another.

Accumulation and separation of membrane-bound proteins using hydrodynamic forces.

The separation of molecules residing in the cell membrane remains a largely unsolved problem in the fields of bioscience and biotechnology. We demonstrate how hydrodynamic forces can be used to both accumulate and separate membrane-bound proteins in their native state. A supported lipid bilayer (SLB) was formed inside a microfluidic channel with the two proteins streptavidin (SA) and cholera toxin (CT) coupled to receptors in the lipid bilayer. The anchored proteins were first driven toward the edge of the lipid bilayer by hydrodynamic forces from a flowing liquid above the SLB, resulting in the accumulation of protein molecules at the edge of the bilayer. After the concentration process, the bulk flow of liquid in the channel was reversed and the accumulated proteins were driven away from the edge of the bilayer. Each type of protein was found to move at a characteristic drift velocity, determined by the frictional coupling between the protein and the lipid bilayer, as well as the size and shape of the protein molecule. Despite having a similar molecular weight, SA and CT could be separated into monomolecular populations using this approach. The method also revealed heterogeneity among the CT molecules, resulting in three subpopulations with different drift velocities. This was tentatively attributed to multivalent interactions between the protein and the monosialoganglioside G(M1) receptors in the lipid bilayer.

Dynamic bioprocessing and microfluidic transport control with smart magnetic nanoparticles in laminar-flow devices.

In the absence of applied forces, the transport of molecules and particulate reagents across laminar flowstreams in microfluidic devices is dominated by the diffusivities of the transported species. While the differential diffusional properties between smaller and larger diagnostic targets and reagents have been exploited for bioseparation and assay applications, there are limitations to methods that depend on these intrinsic size differences. Here a new strategy is described for exploiting the sharply reversible change in size and magnetophoretic mobility of "smart" magnetic nanoparticles (mNPs) to perform bioseparation and target isolation under continuous flow processing conditions. The isolated 5 nm mNPs do not exhibit significant magnetophoretic velocities, but do exhibit high magnetophoretic velocities when aggregated by the action of a pH-responsive polymer coating. A simple external magnet is used to magnetophorese the aggregated mNPs that have captured a diagnostic target from a lower pH laminar flowstream (pH 7.3) to a second higher pH flowstream (pH 8.4) that induces rapid mNP disaggregation. In this second dis-aggregated state and flowstream, the mNPs continue to flow past the magnet rather than being immobilized at the channel surface near the magnet. This stimuli-responsive reagent system has been shown to transfer 81% of a model protein target from an input flowstream to a second flowstream in a continuous flow H-filter device.

Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy.

This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8mum diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology.

A novel, generic and effective method for the rapid purification of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

High-yield production and purification of recombinant T7-tag mature streptavidin in glucose-stressed E. coli.

The overexpression of toxic recombinant proteins is often problematic, leading to either low production levels or inclusion bodies. Streptavidin is no exception and thus the highest production level reported to date for streptavidin is 70 mg/L of functional protein. Herein, we report on the production in Escherichia coli and the purification of a recombinant mature streptavidin bearing a T7-tag. Optimization of critical parameters, including the glucose concentration, the pH and the time of induction as well as the use of BL21(DE3)pLysS cell strain, affords up to 120 mg/L functional streptavidin in soluble form. The yield can be further increased by an osmotic stress during the preculture by adding highly concentrated glucose before the inoculation of the culture medium, thus affording reproducibly 230 mg/L of soluble streptavidin. A single denaturing-renaturing step and affinity chromatography afford highly active tetrameric protein with >3.8/4.0 active sites.