Abstract:
:Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli [Tsumoto et al. (1995) Biochem. Biophys. Res. Commun. 201, 546-551]. Although the expression level of scFvLH is high in vivo, the single chain antibody with the reversed orientation (scFvHL) was synthesized in a very low yield and no active product could be obtained. We report here the synthesis of these two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E. coli expression system in the presence of reduced and oxidized glutathione, protein disulfide isomerase (PDI), and chaperones. In immunological blotting assays, the synthesized scFvs with both arrangements exhibit specific binding activity to the corresponding antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme. scFvLH is synthesized mainly as a product with the expected molecular weight, whereas scFvHL is produced with additional shorter fragments, suggesting that the low yield isolation through the expression in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript. In the cell-free expression of scFv a certain amount of the product is precipitated but in the presence of chaperones the amount of soluble protein increased from 25 to 90% (PDI and chaperones). The overall expression level and the specific biological activity, however, were hardly influenced. The system reported here can provide significant amounts of various scFv fragments regardless of the order of variable regions, including those which are hardly expressed in vivo.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Merk H,Stiege W,Tsumoto K,Kumagai I,Erdmann VAdoi
10.1093/oxfordjournals.jbchem.a022290subject
Has Abstractpub_date
1999-02-01 00:00:00pages
328-33issue
2eissn
0021-924Xissn
1756-2651journal_volume
125pub_type
杂志文章abstract::Mouse epidermal growth factor (EGF) was covalently conjugated with the water-soluble polymer, poly(acrylic acid) (EGF-PAA), or with the water-insoluble polymer, surface-hydrolyzed poly(methyl methacrylate) (EGF-PMMA). Immobilized EGF (EGF-PMMA) stimulated DNA synthesis in Chinese hamster ovary cells overexpressing EGF...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a021616
更新日期:1997-03-01 00:00:00
abstract::I-protein was mixed with myosin before or after myosin filaments were reconstituted. In both cases, I-protein seemed to accelerate the myosin assembly. The binding of I-protein to myosin filaments was tested by sedimentation experiments and SDS-polyacrylamide gel electrophoresis. In a low ionic strength solution at pH...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a122796
更新日期:1989-07-01 00:00:00
abstract::alphaB-Crystallin, a member of the small heat shock protein (sHSP) family, is expressed in various tissues including lens, heart, and skeletal muscle. Previously we identified the gene of HSPB2, another member of the sHSP family, located 1-kb upstream of the alphaB-crystallin gene in a head-to-head manner. In the pres...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvh049
更新日期:2004-03-01 00:00:00
abstract::The activity of biological molecules is often affected by their phosphorylation state. Regulatory phosphorylation operates as a binary switch and is usually controlled by counteracting kinases and phosphatases. However, phosphatidylinositol (PtdIns) has three phosphorylation sites on its inositol ring. The phosphoryla...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvs107
更新日期:2013-01-01 00:00:00
abstract::A major lipid class in the Harderian glands of the Mongolian gerbil was investigated. The IR and 1H-NMR spectra suggested that it was a wax-like compound. Fatty acids present were capric, lauric, myristic, palmitic, stearic, arachidic, and behenic acids in ratios of 3.0%, 16.3%, 16.5%, 29.5%, 5.1%, 16.4%, and 9.8%, re...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134560
更新日期:1983-12-01 00:00:00
abstract::The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with p...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvn039
更新日期:2008-07-01 00:00:00
abstract::Bdellin-KL is a trypsin-plasmin inhibitor from Hirudo nipponia, whose N-terminal sequence was identified as a non-classical Kazal-type. A cDNA clone encoding the inhibitor was isolated by reverse transcription-PCR and 5' rapid amplification of cDNA ends. The cDNA showed an open reading frame of 155 amino acids compris...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a003003
更新日期:2001-09-01 00:00:00
abstract::To clarify the relationship among ryanodine receptor isoforms from skeletal muscles of various vertebrates (alpha- and beta-isoforms with smaller and larger mobilities on SDS-polyacrylamide gel electrophoresis, respectively), we examined the cross-reactivities of those receptors from 13 animal species by Western blot ...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a124636
更新日期:1994-11-01 00:00:00
abstract::We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with r...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a122454
更新日期:1988-08-01 00:00:00
abstract::Lipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about one-te...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a133727
更新日期:1982-02-01 00:00:00
abstract::Two cultured cell lines, called Kan-R1 and Kan-R2, were established from rat hepatic cells by in vitro culture with a hepatocarcinogen, 3-methoxy-4-aminoazobenzene, and examined for the gene expression of cytochrome p450 (p450) isoforms, CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1 and CYP3A2, by the RT-PCR method. It was r...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvg105
更新日期:2003-06-01 00:00:00
abstract::The SoxR protein of Escherichia coli responds to redox signals by activating the transcription of soxS, which encodes another transcription activator that directly stimulates oxidative stress genes. In contrast, Pseudomonas aeruginosa has an open reading frame (ORF) encoding a putative protein homologous to E. coli So...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvh168
更新日期:2004-11-01 00:00:00
abstract::Polynucleotide phosphorylase from Thermus thermophilus catalyzed the addition of short guanylyl blocks from GDP to the 3'-hydroxyl termini of oligonucleotide primers at low temperature in a simple reaction mixture. Polyguanylic acid formation was inhibited at 37 degrees C, but the addition of one or two guanylyl resid...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132660
更新日期:1979-11-01 00:00:00
abstract::Nearly full-length cDNA clones for muscle-type and non-muscle-type aldolase mRNAs were cloned from lambda gt10 cDNA libraries constructed from skeletal muscle and liver mRNAs of lamprey (Entosphenus japonicus). The cDNA-M8 has 2,240 bp carrying an open reading frame of 1,089 bp which encodes 362 amino acids without th...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a124742
更新日期:1995-03-01 00:00:00
abstract::The nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B was determined. PBII is 7.1 kb long and contains 3 exons. PBII exhibits considerable nucleotide sequence homology not only in exons but also in introns with PBI (accession number D89501), the gene whose nucleotide sequence was determined p...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a022620
更新日期:2000-03-01 00:00:00
abstract::The roles of adult worker honeybees change with age; young workers nurse brood by secreting bee milk (royal jelly), and older workers forage for nectar and pollen and process nectar into honey. The electrophoretic profile of worker hypopharyngeal-gland proteins changes with age and/or role. Immunoblotting analysis usi...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a021237
更新日期:1996-02-01 00:00:00
abstract::The present data describe the mechanism of the inhibitory effects of human plasma apolipoproteins A-I and A-II on hydrolysis of triglyceride catalyzed by hepatic triglyceride lipase using a substrate of triolein particles stabilized with gum arabic in vitro. The experimental data could well be described by a model in ...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134000
更新日期:1982-09-01 00:00:00
abstract::We have found that arachidonic (20 : 4) acid is indetectable in phosphatidylinositol and diacyglycerol extracted from Drosophila heads. After careful examinations of the lipid extraction processes and fatty acid detection system (gas-liquid chromatography), we excluded the possibility of the oxidation of polyunsaturat...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a135322
更新日期:1985-09-01 00:00:00
abstract::Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracel...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a003183
更新日期:2002-06-01 00:00:00
abstract::Colicin E5 cleaves tRNAs for Tyr, His, Asn and Asp in their anticodons to abolish protein synthesis in Escherichia coli. We previously showed how its C-terminal RNase domain, E5-CRD, recognizes the anticodon bases but the catalytic mechanism remained to be elucidated. Although the reaction products with 5'-OH and 2',3...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvs077
更新日期:2012-10-01 00:00:00
abstract::The DNA chain elongation mechanisms of mouse DNA polymerases alpha and beta have been analyzed by using denatured DNA with a (dT)n block at the 3'-end as a template in combination with RNA ((rA)12-20)primer. The (rA)12-20-primed DNA product synthesized by DNA polymerase alpha was 3-5 s in size even after prolonged rea...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:
更新日期:1980-12-01 00:00:00
abstract::X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximate...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134682
更新日期:1984-03-01 00:00:00
abstract::Thrombopoietin (TPO) is an important haematopoietic factor in megakaryocytic activities as well as in platelet production. Interleukin 6 (IL-6) can co-stimulate TPO-dependent formation of colony forming unit of megakaryocyte (CFU-Meg) growth which could be responsible for residual platelet formation in TPO-deficient o...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvm111
更新日期:2007-07-01 00:00:00
abstract::CXCL14 is a member of the CXC chemokine family. CXCL14 possesses chemoattractive activity for activated macrophages, immature dendritic cells and natural killer cells. CXCL14-deficient mice do not exhibit clear immune system abnormalities, suggesting that the function of CXCL14 can be compensated for by other chemokin...
journal_title:Journal of biochemistry
pub_type: 杂志文章,评审
doi:10.1093/jb/mvs030
更新日期:2012-05-01 00:00:00
abstract::After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed appr...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:
更新日期:1991-02-01 00:00:00
abstract::Fluorescence resonance energy transfer spectroscopy has been used to study the spatial relationships between probes attached to actin and troponin in the reconstituted skeletal muscle thin filament in the presence and absence of Ca2+ ions. Gln-41 and the nucleotide-binding site of actin were selectively labeled with t...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a021940
更新日期:1998-02-01 00:00:00
abstract::The heavy chain of myosin subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96 K. It was split into 26 K, 50K, and 21 K fragments on trypsin treatment. The effect of actin binding on the susceptibilities of the junctions between 26 K and 50 K and between 50 K and 21 K, and on that of alk...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132708
更新日期:1979-12-01 00:00:00
abstract::Papain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spec...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132289
更新日期:1978-12-01 00:00:00
abstract::The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that p...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/jb/mvm046
更新日期:2007-03-01 00:00:00
abstract::The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar r...
journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a121806
更新日期:1986-10-01 00:00:00