Streptavidin Linker Research Articles

Biotinylation of reducing and non-reducing termini to create plug-and-play polysaccharides

Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.

The Devil Is in the Details: What Do We Really Track in Single-Particle Tracking Experiments of Diffusion in Biological Membranes?

Single-particle tracking (SPT) is an experimental technique that allows one to follow the dynamics of individual molecules in biological membranes with unprecedented precision. Given the importance of lipid and membrane protein diffusion in the formation of nanoscale functional complexes, it is critical to understand what exactly is measured in SPT experiments. To clarify this issue, we employed nanoscale computer simulations designed to match SPT experiments that exploit streptavidin-functionalized Au nanoparticles (AuNPs). The results show that lipid labeling interferes critically with the diffusion process; thus, the diffusion measured in SPT is a far more complex process than what has been assumed. It turns out that the influence of AuNP-based labels on the dynamics of probe lipids includes not only the AuNP-induced viscous drag that is the more significant the larger the NP but, more importantly, also the effects related to the interactions of the streptavidin linker with membrane lipids. Due to these effects, the probe lipid moves in a concerted manner as a complex with the linker protein and numerous unlabeled lipids, which can slow down the motion of the probe by almost an order of magnitude. Furthermore, our simulations show that nonlinker streptavidin tetramers on the AuNP surface are able to interact with the membrane lipids, which could potentially lead to multivalent labeling of the NPs by the probe lipids. Our results further demonstrate that in the submicrosecond time domain the motion of the probe lipid is uncorrelated with the motion of the AuNP, showing that there is a 1 μs limit for the temporal resolution of the SPT technique. However, this limit for the temporal resolution depends on the nanoparticle size and increases rapidly with growing AuNPs. Overall, the results provide a molecular-scale framework to accurately interpret SPT data and to design protocols that minimize label-induced artifacts.

Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR

BackgroundAbscisic acid (ABA) functions as a stress phytohormone in many growth and developmental processes in plants. The ultra-sensitive determination of ABA would help to better understand its vital roles and action mechanisms.ResultsWe report a new sensitive and high throughput quantitative real time immuno-PCR (qIPCR) method based on biotin–avidin linkage system for ABA determination in plants. ABA monoclonal antibody (McAb) coated on the inner surface of PCR well pretreated with glutaraldehyde. The pre-prepared probe complex, including biotinylated McAb, biotinylated DNA and streptavidin linker, was convenient for high throughput operations. Finally, probe DNA was quantified by real-time PCR. The detectable ranges were from 10 to 40 ng/L with a limit of detection (LOD) of 2.5 fg. ABA contents in plant sample were simultaneously analyzed using LC–MS/MS to validate the qIPCR method. The results showed that qIPCR method has good specificity and repeatability with a recovery rate of 96.9%.ConclusionThe qIPCR method is highly sensitive for ABA quantification for actual plant samples with an advantage of using crude extracts instead of intensively purified samples.

Cell-Mediated Proteolytic Release of Growth Factors from Poly(Ethylene Glycol) Matrices.

Engineering in vitro tissue mimetics that resemble the corresponding living tissues requires the 3D arrangement of tissue progenitor cells and their differentiation by localized growth factor (GF) signaling cues. Recent technological advances open a large field of possibilities for the creation of complex GF arrangements. Additionally, cell-instructive biomaterials, which bind GFs by various mechanisms and release them with different kinetics depending on binding affinity, have become available. This paper describes the development of a matrix metalloproteinase (MMP)-degradable streptavidin-based linker module, which allows the release of immobilized GFs from synthetic biomimetic poly(ethylene glycol) hydrogels independently of the hydrogel degradation. The MMP-sensitive streptavidin linker is shown to efficiently bind biotinylated molecules, and as proof of concept, bone morphogenetic protein-2 (BMP-2) delivery via the MMP-degradable linker is used to induce osteogenic differentiation in C2C12 cells and mesenchymal stem cells. The results show a significantly increased net effect of proteolytically releasable BMP-2 in comparison to stably immobilized and soluble BMP-2. This study indicates that a GF delivery system directly responsive to cellular activity can have important implications for the synthesis of tissue mimetics and regenerative medicine, as it can influence the availability, the localization of effects, as well as efficacy of employed GFs.

Characterization and stability of a bioactivated alumina nanomembrane for application in flow devices

Surface modification of alumina nanomembrane (ANP) by theophylline derivative Theo1 by dip-coating was successfully achieved. The hybrid material was characterized by SEM, XPS and confocal microscopy. The presence of Theo1 on the ANP surface was established from salt diffusion and membrane potentials measurements. Changes in the electrical character and transport parameters of the modified sample, associated to Theo1 deposition, were established. Stability of the material under diffusive flow was also ascertained. Moreover, as a result of Theo1 bioactivation, streptavidin linkage to the ANP/Theo1-modified membrane was obtained, which is an indication of streptavidin capture.

Micromotors Powered by Enzyme Catalysis.

Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

Modular poly(ethylene glycol) matrices for the controlled 3D-localized osteogenic differentiation of mesenchymal stem cells.

The in vitro formation of physiologically relevant engineered tissues is still limited by the availability of adequate growth-factor-presenting cell-instructive biomaterials, allowing simultaneous and three-dimensionally localized differentiation of multiple tissue progenitor cells. Together with ever improving technologies such as microfluidics, printing, or lithography, these biomaterials could provide the basis for generating provisional cellular constructs, which can differentiate to form tissue mimetics. Although state-of-the-art biomaterials are endowed with sophisticated modules for time- and space-controlled positioning and release of bioactive molecules, reports on 3D arrangements of differentiation-inducing growth factors are scarce. This paper describes the stable and localized immobilization of biotinylated bioactive molecules to a modular, Factor XIII-cross-linked poly(ethylene glycol) hydrogel platform using a genetically engineered streptavidin linker. Linker incorporation is demonstrated by Western blot, and streptavidin functionality is confirmed by capturing biotinylated alkaline phosphatase (ALP). After optimizing bone morphogenetic protein 2 (BMP-2) biotinylation, streptavidin-modified hydrogels are able to bind and present bioactive BMP-2-biotin. Finally, with this immobilization scheme for BMP-2, the specific osteogenic differentiation of mesenchymal stem cells is demonstrated by inducing ALP expression in confined 3D areas. In future, this platform together with other affinity-based strategies will be useful for the local incorporation of various growth factors for engineering cell-responsive constructs.

In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.

Strategies to Improve Homing of Stem Cells to achieve better Efficacy in Stem Cell Therapy

Stem/progenitor cell based therapeutic approach in our daily routine clinical practice, has been elusive dream in medical sciences and improvement of stem cell homing as one of major challenges in cell therapy programs, has been considered a promising milestone. It has been proved that stem/progenitor cells exhibit a homing response to injured tissues/organs, mediated by interactions of chemokine receptors expressed on the cells and chemokines secreted by the injured tissue. For improvement of directed homing of the cells, many techniques have been developed either to engineer stem/progenitor cells with higher amount of chemokine receptors (stem cell-based strategies) or to modulate the target tissues to release higher level of the corresponding chemokines (target tissue-based strategies). Treatment with chemical compounds, preconditioning of the cells with hypoxia, cytokine and growth factor priming of the cells, genetic modifications, coating of cell surface with antibodies, glycoengineering, coating of stem cells with homing ligands by streptavidin linkers are some of strategies to increase the ability of stem cells to respond to the migratory stimuli. On the other side to modulate target sites to be more attractive for stem cells, some strategies like direct injection of chemokines, direct transfection of the target tissue with chemokine encoding genes, injection of ectopic chemokine expressing cells, application of scaffolds, electrical fields and low level laser have been introduced. These extensive investigations have provided significant potentials to enhance targeted stem/progenitor cells homing. Meanwhile there are still some limitations to apply these findings in clinics. To overcome these limitations, further studies should be aimed, unveiling the molecular and cellular mechanisms underlying endogenous cell trafficking during physiological and pathological events like embryogenesis, inflammation, wound healing, or cancer metastasis. Keywords: Cell therapy, Homing, Stem cells.

Creation of a mouse aortic valve interstitial cell-line

cells releases large amounts of DNA, representing a potential target for drug delivery. Hoechst, a DNA binding molecule used to stain nuclei, can be functionalized to contain reactive groups such as biotin. Insulin-like growth factor-1 (IGF-1), a small molecule with a short half-life is cardioprotective. We hypothesized that conjugating IGF-1 to Hoechst would increase targeting of IGF-1 to injured myocardium. Methods: Hoechst-IGF1 (H-IGF1) was synthesized by binding Hoechst-biotin to biotinylated IGF-1 via a fluorescent streptavidin linker. H-IGF1 cell impermeability and activity were assessed in vitro using cultured macrophages and cardiac progenitor cells (CPCs), respectively. To determine in vivo targeting of H-IGF1 to MI, mice underwent coronary artery ischemia-reperfusion. Six hours following MI, mice were injected intravenously with 70 ng of HIGF1, S-IGF1 (streptavidin bound IGF-1 only) or PBS followed by in vivo imaging. Results: Intact macrophages did not show nuclear staining with HIGF1, while permeabilized cells had an increase in fluorescent Hoechst staining, indicating HIGF1 was cell impermeable but could still bind DNA. H-IGF1 activity, demonstrated by Akt phosphorylation in CPCs was similar to native IGF-1. At 30 minutes post-injection, 3.2% of injected H-IGF1 accumulated in infarcted hearts compared with 1.8% of S-IGF1, with levels unchanged at 2 h post-injection as confirmed by IGF-1 ELISA. Intravenous administration of H-IGF1 pre-bound to exogenous DNA, resulted in reduced IGF-1 targeting suggesting that H-IGF1 localization depended on binding DNA. Conclusion: Our data demonstrate that targeted, intravenous delivery of IGF-1 can be achieved by conjugation to Hoechst-derivatives.

Detecting single-abasic residues within a DNA strand immobilized in a biological nanopore using an integrated CMOS sensor

In this paper, we demonstrate the application of a novel current-measuring sensor (CMS) customized for nanopore applications. The low-noise CMS is fabricated in a 0.35μm CMOS process and is implemented in experiments involving DNA captured in an α-hemolysin (α-HL) nanopore. Specifically, the CMS is used to build a current amplitude map as a function of varying positions of a single-abasic residue within a homopolymer cytosine single-stranded DNA (ssDNA) that is captured and held in the pore. Each ssDNA is immobilized using a biotin-streptavidin linkage. Five different DNA templates are measured and compared: one all-cytosine ssDNA, and four with a single-abasic residue substitution that resides in or near the ~1.5nm aperture of the α-HL channel when the strand is immobilized. The CMOS CMS is shown to resolves the ~5Å displacements of the abasic residue within the varying templates. The demonstration represents an advance in application-specific circuitry that is optimized for small-footprint nanopore applications, including genomic sequencing.

Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains

We report a new approach to probing DNA–protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin–streptavidin linkage withstands ∼20pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65pN; access to this higher force regime is sufficient to probe the responses of protein–DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.

Abstract 365: Targeted Delivery of IGF-1 Following Acute Myocardial Infarction

During acute myocardial infarction (MI) there is excessive necrosis of myocardial cells, leading to the release of large amounts of DNA, representing a potential target for drug delivery. Hoechst, a commonly used molecule for staining nuclei, binds to the minor groove of double-stranded DNA and can be functionalized to contain reactive groups such as free amines, sulfhydryls, and biotin moieties. Insulin-like growth factor-1 (IGF-1), a small molecule with a short half-life is protective immediately following MI, though there is potential for long-term toxicity and off-target effects. Therefore, we hypothesized that conjugating IGF-1 to Hoechst would increase targeting of IGF-1 to the injured myocardium. Hoechst-IGF1 (H-IGF1) was synthesized by binding Hoechst-biotin to biotinylated IGF-1 via a fluorescent streptavidin linker. Intact cells did not show nuclear staining with H-IGF1, while permeabilized cells had a significant increase in blue fluorescent Hoechst staining, indicating H-IGF1 was cell impermeable but could still bind DNA. Activity of H-IGF1 was demonstrated by Akt phosphorylation in cultured cardiac progenitor cells and was similar to native IGF-1. To determine in-vivo targeting of H-IGF1 to MI, mice underwent 30 minutes of coronary artery ligation followed by reperfusion (I/R). Six hours following MI, mice were injected intravenously with 70ng of H-IGF1, S-IGF1 (streptavidin bound IGF-1 only) or PBS followed by in vivo imaging at 30 and 120 minutes post-injection. At 30 minutes post-injection, we found 3.2% (2.2 of 70ng) of the injected dose of H-IGF1 in infarcted hearts compared with 1.8% (1.3 of 70ng) of S-IGF1 (n=5-7; p<0.05). To confirm that targeting of H-IGF1 was dependent on binding DNA, H-IGF1 pre-bound to double-stranded DNA was injected intravenously after I/R. This led to a significant (p<0.05) decrease in targeted IGF-1 levels. IGF-1 levels determined by ELISA 2 hours post-injection demonstrated a similar trend with increased targeting of H-IGF1 compared with S-IGF1 treated mice (4.2±0.6 ng vs. 2.4±0.2 ng; p<0.05). In conclusion, our data demonstrate that intravenous delivery of Hoechst-conjugated IGF-1 increases myocardial targeting. This provides a novel strategy for delivery of growth factors for the treatment of MI.

Synthesis and Biological Evaluation of a Biotinylated Paclitaxel with an Extra-Long Chain Spacer Arm

A biotinylated paclitaxel derivative with an extra-long-chain (LC-LC-Biotin) spacer arm was synthesized using an improved synthetic reaction sequence. The biotinylated paclitaxel analogue retained excellent microtubule stabilizing activity in vitro. Furthermore, it was shown that this analogue can simultaneously engage streptavidin and the binding site on microtubules, making it suitable for localization studies or for the attachment of paclitaxel to solid substrates via a streptavidin linkage.

Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein

An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)3]2+ (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer–protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1pM of PDGF-BB. A calibration curve with a linearity range from 1pM to 10nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.

Electrical detection of sandwich immunoassay on indium tin oxide interdigitated electrodes

Electrical detection of sandwich immunoassay using indium tin oxide (ITO) interdigitated electrodes on a biochip is developed. Gold nanoparticles are utilised as the indication of the antibody/antigen/antibody complex on the electrodes and the gold enhancement process is for signal amplification. The electrical detection of the concentration of target antigen in sandwich immunoassay has been demonstrated. ITO interdigitated electrodes were fabricated on a glass substrate and worked as the detection sites. The capture antibody was first immobilised on the detection sites. The target antigens of certain concentration and detection antibody were applied, respectively. Then, sandwich-like antibody/antigen/antibody complex was constructed on the detection sites. Gold nanoparticles were utilised to bind the sandwich complex through biotin–streptavidin linkage. Then, gold enhancement process was performed for enlarging the size of the gold nanoparticles physically. Therefore a conductive layer was formed across the electrodes. The presence and the concentration of the target antigen can be determined by the resistance value across the electrode measured by an ohmmeter directly. The resistance values showed from 1200 to 700 Ω when the target antigen was in the concentration from 20 to 160 ng/ml. The proposed electrical sandwich immunoassay is simplicity, low cost and miniaturisation; therefore it is possible to be integrated into a microfluidic system and potentially be developed a portable diagnostic device.

An Ultrasound Contrast Agent Targeted to P-Selectin Detects Activated Platelets at Supra-Arterial Shear Flow Conditions

To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewis. Biotinylated polyacrylamide Sialyl Lewis or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10, and 5 dynes/cm2. The ratio of binding and passing microbubbles was defined as capture efficiency. There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewis-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm2. Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm2, capture efficiency was 16.11% on activated platelets versus 21.83% on P-selectin, and, at 40 dynes/cm2, adhesion efficiency was still 3.4% in both groups. There was neither significant adhesion of Sialyl Lewis-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin. Microbubble targeting using sialyl Lewis, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions.

Ultrasound-triggered release of materials entrapped in microbubble–liposome constructs: A tool for targeted drug delivery

We investigated the preparation of ultrasound-triggered drug delivery system, based on a pendant complex of microbubble coated with liposomes. Biotinylated decafluorobutane microbubbles were coated with biotinylated liposomes via a streptavidin linker. Liposomes were prepared incorporating calcein and thrombin. Based on initial concentration of calcein, over 1um3 payload volume per each microbubble–liposome particle was achieved, when 100nm liposomes were used.Insonation of microbubble–liposome pendants in vitro resulted in the complete destruction of microbubbles and triggered release of a significant fraction of the entrapped material. Treatment with 1MHz ultrasound (5 pulses, 100ms, 7MPa peak negative acoustic pressure) resulted in the release of ~30% of entrapped calcein, as estimated by the fluorescence quenching assay.Thrombin release from liposomes complexed with microbubbles (11% of entrapped material) due to ultrasound treatment was estimated by a chromogenic substrate study. Prior to insonation, substrate hydrolysis was at background level. Ultrasound-triggered release of thrombin from the pendant complexes caused an acceleration of blood clotting.

Highly luminescent quantum dots functionalized and their conjugation with IgG

A sol–gel approach including partially removing capping agents, depositing SiO 2 monomers, and growing a SiO 2 shell was developed to generate a bio-compatible functionalization on CdTe quantum dots (QDs). The QDs retained their high photoluminescence (PL) efficiency after coating with a SiO 2 shell (22.5%) by controlling the surface state of the QDs to decrease PL degradation during a sol–gel preparation. Furthermore, the CdTe QDs with a SiO 2 shell were conjugated with IgG using 3-sulfo-N-hydroxysuccinimide or streptavidin–maleimide as linkers. The biotin–streptavidin linker resulted in a high PL efficiency retained (only ∼23% lower than the initial value of the QDs), which is crucial for bio-applications.

Preparation of novel magnetic cellulose microspheres via cellulose binding domain–streptavidin linkage and use for mRNA isolation from eukaryotic cells and tissues

We have developed micron-sized magnetic cellulose microspheres (MCMS), biospecifically coated with streptavidin (SA) using a novel cellulose binding domain and SA fusion protein, to harvest mRNA from eukaryotic cells and tissues. Biospecific connection between SA and MCMS exhibited significant advantages compared with traditional chemical coupling, including convenient and simple preparation, elimination of toxic compounds, and highly efficient extraction of a pure target molecule. To confirm compatibility, mRNA isolated from cell or tissue samples was used for amplification of housekeeping genes ( GAPDH and β-actin) and specific target genes (two fragments V H and V L from the variable fragments of IgG and the gene encoding GLP-1 receptor). Preliminary studies predict that this novel method has potential in future developments in target gene isolation and DNA manipulation.