Specific Binding Activity of Isolated Light Chains of Antibodies

Science ◽  
1967 ◽  
Vol 157 (3789) ◽  
pp. 707-709 ◽  
Author(s):  
T. J. Yoo ◽  
O. A. Roholt ◽  
D. Pressman
Keyword(s):  
Specific Binding ◽  
Binding Activity ◽  
Light Chains

scholarly journals Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

1986 ◽  
Vol 164 (5) ◽  
pp. 1809-1814 ◽  
Author(s):  
V Agnello ◽  
J L Barnes
Keyword(s):  
Rheumatoid Factor ◽  
Primary Structure ◽  
Binding Activity ◽  
Light Chains ◽  
Heat Denaturation ◽  
Antigen Binding ◽  
Rheumatoid Factors ◽  
Simple Method ◽  
Heat Labile

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

scholarly journals Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

2016 ◽  
Vol 72 (9) ◽  
pp. 713-719 ◽  
Author(s):  
Qiujia Chen ◽  
Millie Georgiadis
Keyword(s):  
Dna Binding ◽  
Catalytic Domain ◽  
Specific Binding ◽  
Critical Role ◽  
Binding Activity ◽  
Structural Basis ◽  
Unexpected Finding ◽  
Terminal Inverted Repeat ◽  
Dna Binding Activity ◽  
New Genes

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of theHsmar1transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestralHsmar1terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

Plasmodium vivax MSP-1 peptides have high specific binding activity to human reticulocytes

Vaccine ◽  
2002 ◽  
Vol 20 (9-10) ◽  
pp. 1331-1339 ◽  
Author(s):  
Luis Eduardo Rodrı́guez ◽  
Mauricio Urquiza ◽  
Marisol Ocampo ◽  
Hernando Curtidor ◽  
Jorge Suárez ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Specific Binding ◽  
Binding Activity

Tissue-specific transcription of the mouse alpha-fetoprotein gene promoter is dependent on HNF-1

1989 ◽  
Vol 9 (10) ◽  
pp. 4204-4212
Author(s):  
M H Feuerman ◽  
R Godbout ◽  
R S Ingram ◽  
S M Tilghman
Keyword(s):  
Specific Binding ◽  
Gene Promoter ◽  
Binding Activity ◽  
Alpha Fetoprotein ◽  
Transient Expression Assay ◽  
Band Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Dnase I Footprinting

Previous work identified four upstream cis-acting elements required for tissue-specific expression of the alpha-fetoprotein (AFP) gene: three distal enhancers and a promoter. To further define the role of the promoter in regulating AFP gene expression, segments of the region were tested for the ability to direct transcription of a reporter gene in transient expression assay. Experiments showed that the region within 250 base pairs of the start of transcription was sufficient to confer liver-specific transcription. DNase I footprinting and band shift assays indicated that the region between -130 and -100 was recognized by two factors, one of which was highly sequence specific and found only in hepatoma cells. Competition assays suggested that the liver-specific binding activity was HNF-1, previously identified by its binding to other liver-specific promoters. Mutation of the HNF-1 recognition site at -120 resulted in a significant reduction in transcription in transfection assays, suggesting a biological role for HNF-1 in the regulation of AFP expression.

Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding

1992 ◽  
Vol 12 (5) ◽  
pp. 1940-1949
Author(s):  
A D Keller ◽  
T Maniatis
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Specific Binding ◽  
Regulatory Element ◽  
Zinc Fingers ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Binding Activity ◽  
Necessary And Sufficient ◽  
Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

scholarly journals Colchicine-binding protein of the liver. Its characterization and relation to microtubules.

1975 ◽  
Vol 66 (3) ◽  
pp. 609-620 ◽  
Author(s):  
C Patzelt ◽  
A Singh ◽  
Y L Marchand ◽  
L Orci ◽  
B Jeanrenaud
Keyword(s):  
High Speed ◽  
Specific Binding ◽  
Electrophoretic Analysis ◽  
Binding Activity ◽  
Low Concentrations ◽  
Electrophoretic Behavior ◽  
High Affinity Binding Site ◽  
High Concentrations

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(-3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time-dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.

scholarly journals SPECIFICITY IN THE COMBINATION OF FD FRAGMENTS WITH L CHAINS TO FORM HAPTEN-BINDING SITES

1966 ◽  
Vol 123 (5) ◽  
pp. 921-934 ◽  
Author(s):  
O. A. Roholt ◽  
G. Radzimski ◽  
D. Pressman
Keyword(s):  
Amino Acid ◽  
Propionic Acid ◽  
Binding Sites ◽  
Amino Acid Analysis ◽  
Binding Activity ◽  
Light Chains ◽  
Antibody Activity ◽  
Fc Fragment ◽  
Fab Fragments ◽  
Correct Alignment

In the work reported here we have shown that light chains and Fd fragments can be separated completely in propionic acid and then recombined to form Fab fragments with antibody activity. This experiment indicates that in the recombination a correct alignment of the Fd fragments and the L chains occurs to give a competent antibody site, just as occurs with the recombination of separated heavy and light chains of the antibody; thus the Fc fragment is not required for correct alignment. Fd fragments of antibody alone show very low binding activity toward the specific hapten. As is the case for the combination of heavy and light chains, the combination of Fd fragments and light chains also requires that both components come from antibody from the same rabbit in order to give binding sites. When they are derived from different rabbits producing antibody against the same antigen, they still give Fab fragments as shown by immunoelectrophoresis but do not have competent binding sites. An important observation is that the subunits of the papain digest fractions, FabI and FabII, have the capacity to cross-combine to form active Fab fragments with competent binding sites. FdI from FabI combines with LII chains from FabII to give the composite (FdI-LII) with good binding activity. Likewise, the composite (FdII-LI) has good binding activity. The composites from the two types of antibody molecules yielding different Fab fragments have antibody activity although heretofore these molecules have appeared to be different on the bases of chromatography and amino acid analysis. There is also a preferential combination of the Fd fragments to combine with the correct L fragments to give binding sites since this combination takes preference over the combination of Fd fragments of antibody with light chains of normal globulin (or of light chains of antibody with Fd fragments of normal globulin).

scholarly journals Direct activation of Sex-lethal transcription by the Drosophila runt protein

Development ◽  
1999 ◽  
Vol 126 (1) ◽  
pp. 191-200 ◽  
Author(s):  
S.G. Kramer ◽  
T.M. Jinks ◽  
P. Schedl ◽  
J.P. Gergen
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Specific Binding ◽  
Binding Activity ◽  
Transcriptional Activation Domain ◽  
Downstream Target ◽  
Early Promoter ◽  
Dna Binding Activity ◽  
Sex Lethal

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris

Bioimpacts ◽  
2021 ◽  
Author(s):  
Shirin Movaghar Asareh ◽  
Tahereh Savei ◽  
Sareh Arjmand ◽  
Seyed Omid Ranaei Siadat ◽  
Fataneh Fatemi ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Age Related Macular Degeneration ◽  
Light Chains ◽  
Antigen Binding ◽  
Active Structure ◽  
Age Related ◽  
Fragment Antigen Binding

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

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