Yoichi Kumada

Recent advances in controlled immobilization of proteins onto the surface of the solid substrate and its possible application to proteomics

Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate- and microarray-based techniques have been developed by a number of re- searchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while re- taining structure and native biological function. In this review, we focus on recent advances in immobilization methodol- ogy for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library sys- tems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis.

Screening and Characterization of Affinity Peptide Tags Specific to Polystyrene Supports for the Orientated Immobilization of Proteins

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS‐tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site‐specific immobilization of proteins. The various PS‐tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant‐type glutathione S‐transferases (GSTs) fused with the selected PS‐tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C‐terminus, GST‐PS19 and GST‐PS23, when adsorbed on the PS latex beads had a higher affinity than the wild‐type GST, and the specific remaining activity of the immobilized mutant‐type GSTs was approximately 10 times higher than that of the wild‐type GST. The signal intensity detected for GST‐PS19 and GST‐PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti‐peptide antibody specific for the N‐terminus peptide of GST was much higher than that for the wild‐type GST. These findings indicate that the mutant‐type GSTs fused with the selected peptide tags, PS19 and PS23, could be site‐specifically immobilized on the surface of polystyrene with their N‐terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme‐linked immunosorbent assay (ELISA) systems and protein‐based biochips.

Preparation of monodisperse and size-controlled poly(ethylene glycol) hydrogel nanoparticles using liposome templates.

Liposomes were used as templates to prepare size-controlled and monodisperse poly(ethylene glycol) (PEG) hydrogel nanoparticles. The procedure for the preparation of PEG nanoparticles using liposomes consists of encapsulation of photopolymerizable PEG hydrogel solution into the cavity of the liposomes, extrusion through a membrane with a specific pore size, and photopolymerization of the contents inside the liposomes by UV irradiation. The size distributions of the prepared particles were 1.32+/-0.16 microm (12%), 450+/-62 nm (14%), and 94+/-12 nm (13%) after extrusion through membrane filters with pore sizes of 1 microm, 400 nm, and 100 nm, respectively. With this approach, it is also possible to modify the surface of the hydrogel nanoparticles with various functional groups in a one-step procedure. To functionalize the surface of a PEG nanoparticle, methoxy poly(ethylene glycol)-aldehyde was added as copolymer to the hydrogel-forming components and aldehyde-functionalized PEG nanoparticles could be obtained easily by UV-induced photopolymerization, following conjugation with poly-L-lysine-FITC through amine-aldehyde coupling. The prepared PEG particles showed strong fluorescence from FITC on the edge of the particles using confocal microscopy. The immobilization of biomaterials such as enzymes in hydrogel particles could be performed with loading beta-galactosidases during the hydration step for liposome preparation without additional procedures. The resorufin produced by applying resorufin beta-D-galactopyranoside as the substrate showed the fluorescence under the confocal microscopy.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag)

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli

The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective.

Characterization of polystyrene-binding peptides (PS-tags) for site-specific immobilization of proteins

In this study, we characterized polystyrene-binding peptides (PS-tags) that possess a specific binding affinity for hydrophilic polystyrene (phi-PS) plates. Both the FITC-labeled PS19-1 (RAFIASRRIKRP) and PS19-6 (RIIIRRIRR) peptides showed strong binding affinity for commercially available hydrophilic, but not hydrophobic, PS plates in the presence of the non-ionic surfactant Tween 20. The dissociation constants (K(d)) of the PS19-1 and PS19-6 peptides for the hydrophilic PS-A plate were 169 and 86 nM, respectively, and the K(d) of both peptides increased with the concentration of NaCl or urea. Based on adsorption yield and residual activity of glutathione S-transferase (GST) after fusion with the PS19-6 peptide or its variants, it was found that the basic amino acid in the PS-tags, i.e., Arg was essential for the strong binding affinity of PS-tags in both the peptide and peptide-fused protein forms The aliphatic amino acids in PS19-6 and PS19-6L, such as Ile or Leu, were also effective. Thus, a series of PS-tags that possess this unusual feature, especially the peptides PS19-6 (RIIIRRIRR) and PS19-6L (RLLLRRLRR), are potential candidate affinity peptide tags for site-specific immobilization of proteins onto hydrophilic PS plates, which show potential as solid supports for protein-based biochips.

Efficient immobilization of a ligand antibody with high antigen-binding activity by use of a polystyrene-binding peptide and an intelligent microtiter plate

A method for immobilization of ligand antibody to improve the efficiency and sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) was investigated by the use of anti-TNF-alpha monoclonal antibody chemically conjugated with a polystyrene-binding peptide (PS-tag) and an intelligent microtiter plate with large surface area. We compared both adsorption and antigen-binding activity of the ligand antibody (mAb) and mAb with the PS-tag (mAb-PS-tag) on 3 different PS plates: a hydrophobic PS plate (PS-F-1 plate), a hydrophilic PS plate (PS-A plate), and an intelligent microtiter plate packed with PS beads (PS-E plate). Contact areas of the PS-E plate toward ligand antibody solutions were 7-fold larger than those of conventional PS-F-1 and PS-A plates and consequently, both mAb and mAb-PS-tag were efficiently immobilized on the surface of the PS-E plate due to the significantly enhanced surface area. In particular, when the non-ionic surfactant, Tween20, was present during adsorption of the ligand antibodies, the mAb-PS-tag was site-specifically immobilized on the surface of the PS-E plates and exhibited the highest specific antigen-binding activity. High specific antigen-binding activities were preserved, even though the density of the mAb-PS-tag immobilized on the PS-E plate was reduced with application of smaller volumes. Consequently, the decline in the signal intensities detected from the sandwich ELISA on the PS-E plate was negligible, even when one-fourth the original volume of mAb-PS-tag solution was used to coat the plate. The ligand antibody was immobilized with retention of high antigen-binding activity due to the large surface area of the PS-E plate, specific recognition of the PS-tag, and minimal non-specific interaction of the ligand antibody due to use of Tween 20. Thus, the immobilization method developed in this study resulted in an efficient and highly sensitive sandwich ELISA system that requires smaller amounts of ligand antibodies. This method may be useful for fabrication of protein-based biochips, such as antibody chips.

Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones

The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expression of functional scFvs is increased by co-expression of molecular chaperones. In the present study, we examined the effects of the combination of Trx fusion and molecular chaperone co-expression on the production of a functional scFv. A Trx-fused scFv was obtained in the oxidizing cytoplasm, and co-expression of GroELS and trigger factor had the greatest effect, resulting in a 2.8-fold increase in specific productivity. By contrast, the molecular chaperone DnaKJE had no effect. Moreover, co-expression of DnaKJE with GroELS negated the effects of GroELS. Trx-scFv was purified using a bovine ribonuclease A-coupled Sepharose column, and 2.7 mg/L of purified protein was obtained. Soluble Trx-scFv, expressed and purified as described above, exhibited pH-dependent binding similar to that of the parental full-length antibody. In addition, approximately 80% of the initial binding activity was retained after incubation at 37 degrees C for 2 weeks, indicating that the Trx-scFv fusion protein is quite stable. This strategy might be useful for the preparation of other recombinant scFvs.

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays

The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

High biological activity of a recombinant protein immobilized onto polystyrene

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.