Ikuo Fujii

Evolving catalytic antibodies in a phage-displayed combinatorial library

In vitro affinity maturation for evolving catalytic antibodies has been demonstrated by generating a diverse repertoire of the appropriate complementarity-determining regions on a phage surface. Phage display is followed by a selection based on binding to an altered antigen that was not used at the time of immunization, and provides variants with new catalytic activity and substrate specificity. This library format reduces the time needed to isolate the desired catalytic antibody fragments to under 2 weeks.

In vitro abzyme evolution to optimize antibody recognition for catalysis

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.

An automated system for high-throughput single cell-based breeding

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of α-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

The crystal structure of multidrug-resistance regulator RamR with multiple drugs

RamR is a transcriptional repressor of the gene-encoding RamA protein, which controls the expression of the multidrug efflux system genes acrAB-tolC. RamR is an important multidrug-resistance factor, however, its structure and the identity of the molecules to which it responds have been unknown. Here, we report the crystal structure of RamR in complex with multiple drugs, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. All compounds are found to interact with Phe155 of RamR, and each compound is surrounded by different amino acid residues. Binding of these compounds to RamR reduces its DNA-binding affinity, which results in the increased expression of ramA. Our results reveal significant flexibility in the substrate-recognition region of RamR, which regulates the bacterial efflux participating in multidrug resistance.

Production of monoclonal antibodies for sandwich immunoassay detection of Pacific ciguatoxins

Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.

A lipid-coated catalytic antibody in water-miscible organic solvents

Abstrac A lipid-coated catalytic antibody was prepared by mixing aqueous solutions of antibody and synthetic glycolipids. The lipid-coated catalytic antibody is soluble in organic solvents and showed a remarkable reactivity for hydrolysis of lipophilic esters in a buffer solution containing 20–80% DMSO (dimethyl sulfoxide). DMSO was added to solubilize the lipophilic esters and a native antibody was denatured in the same condition. Michaelis-Menten kinetics showed that the reduced reactivity of a native antibody in DMSO-buffer solutions is due to the largely deduced kcat value but not the small change of Km value.

Ab initio structural comparison between the tetrahedral intermediates and the phosphorous-containing analogues in the ester and amide hydrolysis

Abstract From the viewpoint of hapten design, the molecular structures and charge distributions of the tetrahedral intermediates and their analogues in ester and amide hydrolysis were calculated by ab-initio MP2/6-31+G* level.

Optimazation of the loop length for folding of a helix-loop-helix peptide

Abstract We have designed and synthesized compact helix-loop-helix peptides as a scaffold for conformationally defined peptide mimetics. Circular dichroism and sedimentation equilibrium studies suggested that the connection between the two helical segments significantly affected the structural formation. The loop length corresponding to seven glycine residues (approximately 25 A) was found to be most suitable for the intrachain packing of the α-helices.

Development of a monoclonal antibody against the left wing of ciguatoxin CTX1B: Thiol strategy and detection using a sandwich ELISA

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL.