Functional glass surface displaying a glutamyl donor substrate for transglutaminase-mediated protein immobilization

A chemically modified glass surface displaying a glutamyl donor substrate peptide (Z-QG) was developed for microbial transglutaminase (MTG)-mediated immobilization of recombinant proteins tagged with an MTG-reactive lysine-containing substrate peptide (K-tag). To evaluate the surface modification conditions affecting the enzymatic protein immobilization, we employed an amino-modified 96-well glass plate as a base and prepared three types of glass surfaces displaying Z-QG. Validation of the Z-QG modified glass surfaces with recombinant enhanced green fluorescent proteins revealed that the insertion of a di(ethylene glycol) linker between the terminal Z-QG moiety and the base not only enhances enzymatic protein immobilization efficiency but also decreases nonselective protein adsorption. A bacterial alkaline phosphatase fused with a K-tag at the N terminus was also successfully immobilized to the designed glass surface, suggesting that the chemically modified glass surface displaying a spatially controlled glutamyl donor substrate is a potential platform for MTG-mediated fabrication of protein-based solid biomaterials.     

Immobilization and clustering of structurally defined oligosaccharides for sugar chips: an improved method for surface plasmon resonance analysis of protein-carbohydrate interactions

Oligosaccharides are increasingly being recognized as important partners in receptor-ligand binding and cellular signaling. Surface plasmon resonance (SPR) is a very powerful tool for the real-time study of the specific interactions between biological molecules. We report here an advanced method for the immobilization of oligosaccharides in clustered structures for SPR and their application to the analysis of heparin-protein interactions. Reductive amination reactions and linker molecules were designed and optimized. Using mono-, tri-, or tetravalent linker compounds, we incorporated synthetic structurally defined disaccharide units of heparin and immobilized them as ligands for SPR. Their binding to an important hemostatic protein, von Willebrand factor (vWf), and its known heparin-binding domain was quantitatively analyzed. These multivalent ligand conjugates exhibited reproducible binding behavior, with consistency of the surface conditions of the SPR chip. This novel technique for oligosaccharide immobilization in SPR studies is accurate, specific, and easily applicable to both synthetic and naturally derived oligosaccharides.     

SPR imaging-based monitoring of caspase-3 activation

The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.     

A chemically modified glass surface that facilitates transglutaminase-mediated protein immobilization

An amino-modified glass surface for enzymatic protein immobilization by microbial transglutaminase (MTG) was developed. Diamine substrates with secondary amino groups in the linker moiety, like triethylenetetramine (TETA), exhibited at most a 2-fold higher reactivity in the MTG-catalyzed reaction compared to those with the alkyl linker. A 96-well glass plate was subsequently modified with selected diamine substrates. Validation of the modified surface by enzymatic immobilization of enhanced green fluorescent protein tagged with a glutamine donor-substrate peptide (LLQG) of MTG revealed that the protein loading onto the TETA-modified glass surface was approximately 15-fold higher than that on the unmodified one.     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

Carbopeptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template.

Multifunctional, topological template molecules such as linear and cyclic peptides have been used for the attachment of peptide strands to form novel protein models of, for example, 4-alpha-helix bundles. The concept of carbohydrates as templates for de novo design of potential protein models has been previously described and these novel chimeric compounds were termed carbopeptides. Here, a second generation strategy in which carbopeptides are synthesized by chemoselective ligation of a peptide aldehyde to an aminooxy-functionalized alpha-D-galactopyranoside is described. This template was prepared by per-O-acylation of methyl alpha-D-galactopyranoside with N,N-Boc2-aminooxyacetic acid to form a tetra-functionalized template, followed by treatment with TFA-CH2Cl2 to release the aminooxy functionality. The peptide aldehydes Fmoc-Ser-Gly-Gly-H and H-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H were synthesized by a BAL strategy. Four identical copies of peptide aldehyde were smoothly attached to the template by chemoselective ligation to form a 2.1 and a 2.9 kDa carbopeptide, respectively.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.     

End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation

We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM(456)) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM(456) derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM(456) was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM(456) was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM(456) was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity.     

A generic expression system to produce proteins that co-assemble with alkane thiol SAM

Surface biology aims to observe and control biological processes by combining bio-, surface, and physical chemistry. Self-assembled monolayers (SAM) on gold surfaces have provided excellent methods for nanoscale surface preparation for such studies. However, extension of this work requires the specific immobilization of whole protein domains and the direct incorporation of recombinant proteins into SAM is still problematic. In this study a short random coil peptide has been designed to insert into thioalkane layers by formation of a hydrophobic helix. Surface plasmon resonance (SPR) studies show that specific immobilization via the internal cysteine is achieved. Addition of the peptide sequence to the terminus of a protein at the genetic level enables the production of a range of recombinant fusion-proteins with good yield. SPR shows that the proteins display the same gold-binding behavior as the peptide. It is shown that cell growth control can be achieved by printing the proteins using soft lithography with subsequent infilling with thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.     

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.     

Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. K _D values of ~300 μM were determined for both strategies at pH 7.4. This compared with a K _D value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.     

Nanoscale fabrication of protein A on self-assembled monolayer and its application to surface plasmon resonance immunosensor

The fabrication of protein A film on self-assembled monolayer was done for the construction of immunosensor using surface plasmon resonance (SPR) measurement. The layer of heterobifunctional linker, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was self-assembled on the gold (Au) surface. Due to the succinimidyl functional group in SPDP to be reacted with amine (NH₂) group of protein A, the covalent immobilization of protein A was subsequently induced toward Au surface. The characteristics of film formation were investigated using SPR with respect to the various concentrations of SPDP and protein A. The optimal concentration for the film formation was found to be 0.1 mg/mL of SPDP and 0.1 mg/mL of protein A, respectively. The surface topography of protein A layer using atomic force microscopy showed that the heteromolecular layer was formed successfully. The antibody, anti-bovine serum albumin (BSA), was immobilized onto protein A layer, and the fabricated antibody layer was applied for the detection of BSA. The extent of BSA–antibody binding was measured using SPR and its lower detection limit of BSA was 100 pM.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

Proteomics evaluation of chemically cleavable activity-based probes

Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.     

Covalent immobilization of acid phosphatase on amorphous AlPO4 support

Covalent attachment of acid phosphatase enzyme, AP, on the surface of amorphous AlPO₄, used as inorganic support, was studied. Immobilization of the enzyme was carried out by the ε-amino group of lysine residues through an aromatic Schiff's-base (linker A), as well as through an `azo' linkage to a p-OH-benzene group of tyrosine residues of the proteins (linker B). Activation of the supports in both cases was developed through the reaction of appropriate molecules with support surface –OH groups. The enzymatic activities in the 1-naphthyl phosphate hydrolysis of native, the different immobilized AP systems, and the filtrates, were obtained by a spectrophotometric method. According to the results, immobilization through linker A gave E_imm=99% while the residual activity, E_res, at different temperatures was in the range 0.2–0.8%. On the other hand, in the immobilization by linker B, through a diazonium salt, E_imm was in the range 35–46%, but residual and specific activity values, E_res and E_spe, were between 19% and 46%. Thus, instead of linker A was more effective in the enzyme immobilization, the highest enzymatic activity after immobilization was obtained with linker B because with linker A a strong deactivation was developed.     

Design and characterization of a maltose binding protein-linked growth factor for matrix engineering

The maltose-binding protein (MBP), which possesses a large number of exposed hydrophobic zones, can be used as a link for the immobilization of growth factors. The amount of immobilized MBP-vascular endothelial growth factors (VEGFs) for polystyrene surface was increased with respect to increasing protein, showing 1019 ng/cm² at 100 μg protein/ml. The phosphorylation of VEGF receptors in the MBP-VEGF stimulated HEK293/KDR cells as depicted from western blot analysis. Cell adhesion to a MBP-VEGF immobilized surface was mediated by the VEGF-VEGFR interaction. These results suggest that MBP-VEGFs are active and a MBP immobilization system can then anchor various bioactive proteins to hydrophobic surfaces.     

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.     

Study on orientation of immunoglobulin G on protein G layer

A comparative study of immunoglobulin G (IgG) immobilization was performed, both on a thiolated protein G layer, where this immobilization was due to affinity binding with an Fc fragment of IgG, and on 11-mercaptoundecanoic acid (11-MUA), where the immobilization was due to chemical bonding. The change of IgG layer formation on the two base layers as a function of the IgG concentration was investigated by surface plasmon resonance (SPR), atomic force microscopy (AFM) in a non-contact mode, and spectroscopic ellipsometry (SE). It was observed that the IgG layer was immobilized more evenly on the thiolated protein G layer than on the 11-MUA layer, based on the SPR measurements. The surface topology analysis by AFM indicated that the IgG layer was immobilized on the protein G layer according to the envelope profile of the base layer. Based on the SE analysis, it was determined that the IgG layer thickness on the thiolated protein G layer increased with increasing IgG concentration. Based on the above analyses, the scheme for orientation of IgG immobilized on the thiolated protein G layer was proposed.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.