Bioassisted capture and release of nanoparticles on nanolithographed ZnO films.

Using an artificial peptide library, we have identified a peptide that has strict selective affinity for ZnO surfaces. The binding affinity of the peptide on the ZnO surface can be controlled simply through changes in phosphate concentration at constant pH and temperature. In this study, we functionalized inorganic nanoparticles by orderly conjugating ZnO-binding peptides (ZnOBPs) on the surface of cadmium selenide (CdSe) nanoparticles and performed spontaneous and reversible nanopatterning of ZnOBP-displayed nanoparticles on lithographed ZnO films. Conjugation of ZnOBPs on CdSe nanoparticles caused spontaneous adsorption of the nanoparticles on a ZnO film, and fluorescence and cathodoluminescence images clearly showed specific adsorption of nanoparticles on the ZnO films lithographed on nano- and micrometer scales. The selectively bound nanoparticles on ZnO films were completely released by changing the phosphate concentration in solution; such release did not require heat or mechanical applications. Repeated capture and release of nanoparticles were achieved on the micrometer scale. Our results show the potential of material-binding peptides for nanopatterning and dynamic microarrays.

Open sandwich ELISA: a novel immunoassay based on the interchain interaction of antibody variable region.

We describe an immunoassay that is based on the interchain interaction of separated VL and VH chains from a single chain antibody variable region. In the presence of antigen, the chains reassociate. VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 were immobilized on microtiter plates. Samples were coincubated with an M13-displayed VH chain, and assayed with peroxidase-labeled anti-M13 antibody. Signal was detected in direct proportion to the amount of HEL in the sample. Wide dynamic range with < 15 ng/ml sensitivity was attained.

Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides.

We introduced 4-thio- ((4S)U), 2-thio- ((2S)U), 4- O -methyluridine ((4Me)U) and cytidine substitutions for U+2, which is an important base for cleavage in a substrate RNA. Oligonucleotides containing 4-thio- and 4- O -methyluridine were prepared by a new convenient post-synthetic modification method using a 4- O - p -nitrophenyl-uridine derivative. A hairpin ribozyme cleaved the substrate RNA with either C+2, (4S)U+2 or (4Me)U+2 at approximately 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a (2S)U+2 was cleaved with the same activity as the natural substrate. These results suggest that the O4 of U+2 is involved in hydrogen bonding at loop A, but the O2 of U+2 is not recognized by the active residues. Circular dichroism data of the ribozymes containing (4S)U+2 and (2S)U+2, as well as the susceptibility of the thiocarbonyl group to hydrogen peroxide, suggest that a conformational change of U+2 occurs during the domain docking in the cleavage reaction. We propose here the conformational change of U+2 from the ground state to the active molecule during the reaction.

Enzymatic properties of an acid carboxypeptidase from Aspergillus niger var. macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) isolated from Aspergillus niger var. macrosporus was investigated in regard to its kinetic parameters for two synthetic substrates. The optimum pH of peptidase activity toward Z-Glu-Tyr-OH was pH 3.0 Km and kappa cat values were 4.0 . 10(-3) M and 270 s-1 at pH 3.0 and 30 degrees C. The optimal pH of esterase activity toward Bz-Arg-OEt was pH 5.2 Km and kappa cat for esterolytic activity were 6.1 . 10(-4) M and 1500 s-1 at pH 5.0 and 30 degrees C. The enzyme released expected amino acids sequentially from the carboxyl ends of S-beta-aminoethylated ribonuclease A and the B-chain of oxidized insulin, demonstrating carboxypeptidase activity of the enzyme. The enzyme was inactivated by diisopropylphosphorofluoridate and phenyl-methanesulfonyl fluoride. In the reaction with [14C]diisopropylphosphorofluoridate, the amount of [14C]diisopropylphosphoryl group incorporated into the enzyme in complete inactivation was estimated as 2 mol/mol enzyme.

Isolation, purification and some chemical properties of an acid carboxypeptidase from Aspergillus niger var. Macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) was purified to a homogeneous state from the water extracts of Koji cultures of Aspergillus niger var. macrosporus. The molecular weight of the enzyme was determined to e 136 000 by sedimentation equilibrium method. The denatured specimen of the enzyme exhibited a molecular weight of 60 000 in the sedimentation equilibrium in 6 M guanidinium chloride, suggesting that the native enzyme is composed of two identical subunits. However, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the enzyme showed an anomalous Ferguson plot, which may account for the inconsistent values of apparent molecular weights obtained by this method. The acid carboxypeptidase was found to be an acidic glycoprotein (pI, 4.1), composed of 955 amino acid, 140 mannose, 14 galactose and 30 glucosamine residues/molecule.

Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene.

To elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E. coli. This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid. When the resultant plasmid pMKSI161-9 was introduced into E. coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis. When introduced into 'leaky' E. coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria. NH2-terminal sequencing analysis of the SSI purified from E. coli JM105 indicated two processing sites, Ala(-4)/Ala(-3)-Pro(-2)-Gly and Ala(-4)-Ala-3/Pro(-2)-Gly-1, of pre-SII. These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66. The inhibitor activity of the processed protein toward subtilisin BPN' was almost the same as that of authentic SSI.

Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans.

Streptomyces are bacteria with a very high chromosomal G+C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu80: CTG-CTC to TTA-TTA, Lys89: AAG to AAA, Ser108-Ser109: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in the case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNA(Leu) (UUA), a product of bldA which has been reported to be expressed only during the late stage of growth.

Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode.

Trp62 in hen egg-white lysozyme has general features observed in protein-carbohydrate interactions, a stacking interaction toward nonpolar surface of the substrate sugar residue B and a hydrogen bonding network with the residue C. Our previous report (I. Kumagai, K. Maenaka, F. Sunada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) showed that the substitution of Trp62 changed the substrate binding modes; especially, the Trp62His mutant exhibited the drastic change of the binding mode and preferred to a minor binding mode of the wild-type enzyme. In order to clarify the relationship between functional and structural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, (GlcNAc)3, by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild-type. His62 shared almost the same plane as the indole ring of Trp62 of the wild-type. Although the (GlcNAc)3 molecule which is an inhibitor against the wild-type lysozyme was cocrystallized, the Trp62His mutant did not put it in the sites A-B-C but hydrolyzed it as a substrate. One of the products, (GlcNAc)2, whose reducing end is alpha-anomer, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anomer product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interactions between His62 and the sugar residues B and C.

Synthesis of alpha-helix-forming peptides by gene engineering methods and their characterization by circular dichroism spectra measurements.

Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.

Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Requirement for a disulfide bridge near the reactive site of protease inhibitor SSI (Streptomyces subtilisin inhibitor) for its inhibitory action.

The problem of why serine protease inhibitors having substrate-like structure around the reactive site are not degraded by the cognate protease has prompted us to investigate the structural requirements in Streptomyces subtilisin inhibitor (SSI) for its inhibitory action. We removed the disulfide bridge between Cys71 and Cys101 near the reactive site by oligonucleotide-directed mutagenesis, replacing both Cys residues with Ser residues. Inhibitory activity of the mutated SSI toward subtilisin BPN' was initially potent, but decreased remarkably with increasing incubation time after mixing (temporary inhibition), due to degradation of the mutated SSI by subtilisin via a specific intermediate with a nick at the reactive site (Met73-Val74). Binding affinity of subtilisin for the mutated SSI was reduced by more than 10(3)-fold, and the mutated SSI showed a 20 deg.C decrease in melting temperature, which probably mainly reflects the destruction of the region of alpha-helix containing Cys101. These results imply that the susceptibility of the mutated SSI to protease, and the irreversibility of the peptide bond cleavage at the reactive site, result from increased flexibility around the reactive site in the complex of the disulfide-bond-removed SSI with the protease, demonstrating the requirement of conformational rigidity around the reactive site of the inhibitor for its native inhibitory action.

Dissection of protein-carbohydrate interactions in mutant hen egg-white lysozyme complexes and their hydrolytic activity.

Trp62 in the binding subsite B of hen egg-white lysozyme shows general features often observed in protein-carbohydrate interactions including a stacking interaction and a hydrogen bonding network with water molecules. A previous report by our group showed that the perturbation of these interactions by substitution of Trp62 with tyrosine or phenylalanine affects the substrate binding modes and also enhances the hydrolytic activity. In order to elucidate the relationship between structural and functional changes of these protein-carbohydrate interactions, the Trp62Tyr and Trp62Phe mutants complexed with the substrate analogue, (GlcNAc)3, were analyzed at 1.8 A resolution by X-ray crystallography. The overall structures of the mutant enzymes are indistinguishable from that of the wild type enzyme. Although the wild-type enzyme binds (GlcNAc)3 in only one binding mode (A-B-C), the Trp62Tyr mutant binds (GlcNAc)3 in two binding modes (A-B-C, B-C-D) and the Trp62Phe mutant has an even weaker binding mode. The aromatic rings of Tyr62 and Phe62 maintain their interactions with the carbohydrate molecules, but make fewer stacking interactions with the GlcNAc in the B site than the wild-type enzyme does. The hydroxyl group of Tyr62 interacts weakly with a water molecule which mediates hydrogen bonding in the GlcNAc residues in the B and C sites. The C-6 hydroxyl group of the GlcNAc residue in the C site rotates around the C-5-C-6 bond to complete the hydrogen bond network in the Trp62Tyr mutant-(GlcNAc)3 complex. On the other hand, this hydrogen bonding network does not form in the Trp62Phe mutant-(GlcNAc)3. In addition to these structural studies, the kinetic parameters of the hydrolysis of 4-methylumbelliferyl N-acetyl-chitotriose, ((GlcNAc)3-MeU), have been determined in order to further characterize the enzymatic properties of these mutant lysozymes. This demonstrates that the modulation of the hydrogen bonding network, including the flexible part of the carbohydrate and water molecules and/or the slight reduction of stacking interaction in the B site, alters the binding mode toward the carbohydrate and induces an enhancement of the hydrolytic activity.

Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli.

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.

Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans.

A secretory production system for the Fv domain of a monoclonal antibody (mAb) against hen egg-white lysozyme (HEL) was established in Streptomyces lividans using a chemically synthesized gene. The synthetic DNAs encoding the Fv fragments (VH and VL) of the anti-HEL mAb, HyHEL10, were fused to DNA encoding the signal peptide of Streptomyces subtilisin inhibitor (SPssi) in an SPssi::VH-SPssi::VL dicistronic arrangement. The genes were expressed under the control of the ssi promoter using S. lividans as host. Each Fv fragment was accurately processed and secreted into the growth medium. No inclusion bodies were produced. The Fv fragments were isolated from culture supernatant by a two-step purification (affinity chromatography and gel filtration) with a high yield (approx. 1 microgram/ml). Purified Fv fragments bound to HEL specifically, and completely inhibited the catalytic activity of HEL at a molar ratio of 1.25 for Fv vs. HEL.

Analysis of transcriptional control regions in the Streptomyces subtilisin-inhibitor-encoding gene.

A transcript, of about 650 nucleotides (nt), from the Streptomyces subtilisin-inhibitor-encoding gene (ssi) was identified by Northern hybridization analysis in both the original strain, S. albogriseolus S-3253, and the transformant, S. lividans 66, carrying an expression plasmid with the cloned ssi gene, pJS1. These results were quite consistent with the analysis of the major transcriptional start point (tsp; at nt 429) by primer extension experiments and the transcriptional end point (at nt 1065) by S1 nuclease mapping of the ssi gene. Deletion experiments on the 5'-flanking region of the major tsp suggested that two promoter sequences control the expression of ssi. The more proximal of these putative promoters appears to be homologous to the -45 to -25 region of the ctc promoter in Bacillus subtilis and includes a direct repeat in the -10 region.

Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue.

Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.

Unusual genetic codes and a novel gene structure for tRNA(AGYSer) in starfish mitochondrial DNA.

The nucleotide sequence of a 3849-bp fragment of starfish mitochondrial genome was determined. The genes for NADH dehydrogenase subunits 3, 4, 5, and COIII, and three kinds of (tRNA(UCNSer), tRNA(His), and tRNA(AGYSer) were identified by comparing with the genes of other animal mitochondria so far elucidated. The gene arrangement of starfish mitochondrial genome was different from those of vertebrate and insect mitochondrial genomes. Comparison of the protein-encoding nucleotide sequences of starfish mitochondria with those of other animal mitochondria suggested a unique genetic code in starfish mitochondrial genome; both AGA and AGG (arginine in the universal code) code for serine, AUA (isoleucine in the universal code but methionine in most mitochondrial systems) for isoleucine, and AAA (lysine) for asparagine. It was also inferred that these AGA and AGG codons are decoded by serine tRNA(AGYSer) originally corresponding to AGC and AGU codons. This situation is similar to the case of Drosophila mitochondrial genome. Variations in the use of AGA and AGG codons were discussed on the basis of the evolution of animals and decoding capacity of various tRNA(AGYSer) species possessing different sizes of the dihydrouridine (D) arm.

Contribution of salt bridge in the protease inhibitor SSI (Streptomyces subtilisin inhibitor) to its inhibitory action.

The tertiary structure of proteinaceous protease inhibitors is considered to be maintained by various interactions in the molecule that prevent degradation by protease. In this study, the Arg29 of Streptomyces subtilisin inhibitor (SSI) forming a salt bridge with the carboxyl group of carboxyl-terminal Phe113 was replaced with Ala, Met or Lys by cassette mutagenesis to clarify the role of Arg29 in the function of SSI. The inhibitory activity of each mutated SSI decreased with increasing incubation time after mixing with subtilisin, indicating that the SSI was changed into a temporary inhibitor upon mutation. This decrease was shown by SDS polyacrylamide gel electrophoresis to be due to cooperative degradation of the mutated SSI by subtilisin. In addition, the denaturation temperature of the Ala or Met mutant was decreased by ten degrees and that of the Lys mutant by 1.5 degrees, suggesting that the destabilization of SSI may be related to its temporary inhibition. Thus, interaction in the protease inhibitor molecule for maintaining the tertiary structure, such as that of Arg29 in SSI, was shown to be required for the inhibitory action.

Functional Fv fragment of an antibody specific for CD28: Fv-mediated co-stimulation of T cells.

The most predominant co-stimulation pathway, which is critical for T cell activation and proliferation, is the CD28-B7 pathway. The anti-CD28 monoclonal antibody (mAb) also provides a co-stimulatory signal to T cells. In order to construct a functional Fv fragment (complex of VH and VL domains) of anti-CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli. The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG. The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti-CD3 or anti-CD2 mAb and enhanced anti-tumor activity of anti-MUC1x(anti)-CD3 bispecific antibody when tested with lymphokine-activated killer cells with T cell phenotype. Thus, the anti-CD28 Fv fragment will be promising not only for the study of co-stimulation, but also for cancer immunotherapy.

Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent--application to a human single-chain Fv fragment.

An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed. Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments.