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1.
Cell Death Dis ; 3: e295, 2012 Apr 12.
Article in English | MEDLINE | ID: mdl-22495350

ABSTRACT

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC(50) ∼2 × 10(-12) M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance.


Subject(s)
Colonic Neoplasms/drug therapy , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor , Dimerization , ErbB Receptors/genetics , ErbB Receptors/metabolism , HEK293 Cells , Hep G2 Cells , Humans , Jurkat Cells , Mice , Mice, Nude , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/therapeutic use , Transplantation, Heterologous
2.
Protein Eng ; 14(10): 815-23, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11739902

ABSTRACT

Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies.


Subject(s)
Antibodies, Bispecific/biosynthesis , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/immunology , Antigen-Antibody Reactions , Antigens/immunology , Dimerization , Enzyme-Linked Immunosorbent Assay , Oligonucleotides , Peptide Library , beta-Galactosidase
3.
Mol Ther ; 3(6): 882-91, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11407902

ABSTRACT

The use of adenoviruses for antivascular cancer gene therapy is limited by their low transduction efficiency for endothelial cells. We have developed a recombinant bispecific antibody as a molecular bridge, linking the adenovirus capsid to the endothelial cell surface protein endoglin, for vascular targeting of adenoviruses. Endoglin (CD105), a component of the transforming growth factor beta receptor complex, represents a promising target for antivascular cancer therapy. Endoglin is expressed predominantly on endothelial cells and is upregulated in angiogenic areas of tumors. We isolated single-chain Fv fragments directed against human endoglin from a human semisynthetic antibody library. One of the isolated scFv fragments (scFv C4) bound specifically to various proliferating primary endothelial cells or cell lines including HUVEC, HDMEC, HMVEC, and HMEC. ScFv C4 was therefore used to construct a bispecific single-chain diabody directed against endoglin and the adenovirus fiber knob domain (scDb EDG-Ad). This bispecific molecule mediated enhanced and selective adenovirus transduction of HUVECs, which was independent from binding to the coxsackievirus and adenovirus receptor (CAR) and alpha(v)-integrins. Thus, adenovirus infection was redirected to a new cellular receptor (CD105) and cell entry pathway. These results demonstrate the utility of bispecific single-chain diabodies, which can be produced in large quantities in bacteria, for the retargeting of adenoviruses in cancer gene therapy.


Subject(s)
Adenoviridae/genetics , Antibodies, Bispecific/genetics , Genetic Therapy/methods , Vascular Cell Adhesion Molecule-1/genetics , Adenoviridae/immunology , Antibodies, Viral/genetics , Antigens, CD , Base Sequence , Blotting, Western , Cells, Cultured/metabolism , Cloning, Molecular , Endoglin , Endothelium, Vascular/immunology , Endothelium, Vascular/physiology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Targeting/methods , Genetic Vectors , Humans , Immunoblotting , Immunoglobulin Fragments/immunology , Immunoglobulin Variable Region/immunology , Molecular Sequence Data , Peptide Library , Peptides, Cyclic/metabolism , Receptors, Cell Surface , Recombinant Proteins/metabolism , Umbilical Veins/physiology , Vascular Cell Adhesion Molecule-1/immunology
4.
Br J Cancer ; 81(8): 1269-73, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10604721

ABSTRACT

Angiostatin, a potent inhibitor of angiogenesis, tumour growth and metastasis, is a biologically active fragment of plasminogen, containing the kringle domains 1-4. It is generated from plasminogen by limited proteolysis. We show that prostate-specific antigen (PSA), a serine proteinase secreted by human prostate and human prostate cancer cells, is able to convert Lys-plasminogen to biologically active angiostatin-like fragments, containing kringles 1-4, by limited proteolysis of peptide bond Glu439-Ala440 in vitro. In an in vitro morphogenesis assay, the purified angiostatin-like fragments inhibited proliferation and tubular formation of human umbilical vein endothelial cells with the same efficacy as angiostatin. This finding might help to understand growth characteristics of prostate cancer, which usually has low microvessel density and slow proliferation.


Subject(s)
Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Plasminogen/chemistry , Prostate-Specific Antigen/chemistry , Amino Acid Sequence , Angiostatins , Cells, Cultured , Humans , Male , Peptide Fragments/isolation & purification
5.
J Immunol Methods ; 226(1-2): 179-88, 1999 Jun 24.
Article in English | MEDLINE | ID: mdl-10410983

ABSTRACT

Intracellularly expressed antibody fragments have found various applications in therapy by virtue of their ability to inhibit the function of cellular proteins or interfere with subcellular trafficking. Bivalent antibody fragments might further improve this inhibitory potential by increasing the functional affinity and bispecific antibody fragments may also be useful for the intracellular retargeting of molecules. Here, we have evaluated the functional expression of intracellular diabodies. A previously constructed secreted bispecific single-chain diabody directed against carcinoembryonic antigen and Escherichia coli beta-galactosidase was modified for subcellular targeting to the cell surface membrane, endoplasmic reticulum, mitochondria, cytoplasm, and nucleus. Subcellular localisation was analysed by immunofluorescence, and the assembly of functional antibodies was analysed by binding of beta-galactosidase to the antibody fragment and subsequent substrate conversion. Bispecific single-chain diabodies could be directed to all subcellular compartments analysed. However, functional assembly was only observed for single-chain diabodies retained in the endoplasmic reticulum or displayed in the cell membrane while no antigen binding activity was seen with diabodies directed to the cytoplasm, nucleus, or mitochondria. The results demonstrate the functional expression of bispecific recombinant antibody fragments in the secretory pathway and integration into the plasma membrane of mammalian cells.


Subject(s)
Antibodies, Bispecific/biosynthesis , Carcinoembryonic Antigen/immunology , Immunoglobulin Fragments/biosynthesis , beta-Galactosidase/immunology , 3T3 Cells , Amino Acid Sequence , Animals , Antibodies, Bispecific/immunology , Cell Line , Cell Membrane/metabolism , Escherichia coli/enzymology , Humans , Immunoglobulin Fragments/immunology , Intracellular Fluid/metabolism , Mice , Molecular Sequence Data , Subcellular Fractions
6.
FEBS Lett ; 454(1-2): 90-4, 1999 Jul 02.
Article in English | MEDLINE | ID: mdl-10413102

ABSTRACT

Although bispecific IgG molecules have been successfully applied for antibody-mediated immunotherapy of tumours, applicability is hampered by the difficulties associated with their generation. In the present study, we have used a bispecific single-chain diabody (scDb) directed against carcinoembryonic antigen and Escherichia coli beta-galactosidase as a model to generate bispecific IgG-like antibody molecules. We show that the fusion of this single-chain diabody to the Fc (scDb-Fc) or CH3 (scDb-CH3) region of the human immunoglobulin gamma1 chain results in the expression of dimeric fusion proteins exhibiting four functional antigen binding sites with increased functional affinity. This strategy represents a new and convenient way to generate IgG-like multivalent and bispecific molecules that are efficiently secreted from mammalian cells.


Subject(s)
Antibodies, Bispecific/immunology , Immunoglobulin Constant Regions/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulins/immunology , Antibody Affinity , Cell Line , Chromatography, Gel , Dose-Response Relationship, Immunologic , Humans , Models, Biological
7.
Thromb Res ; 92(1): 33-41, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9783672

ABSTRACT

Engineering of recombinant coagulation factor X variants, which can be activated by tumor-associated proteinases may lead to the development of new therapeutic molecules. However, the evaluation of such variants requires an appropriate animal model. Therefore, we isolated the complete coding sequence of mouse coagulation factor X from mouse liver cDNA by polymerase chain reaction. The deduced amino acid sequence codes for a prepro protein of 481 amino acids homologous to factor X sequences from various species. Recombinant mouse factor X was expressed in human embryonic kidney cells and secreted into cell culture supernatant as zymogen, which could be converted to catalytically active factor Xa by Russell's viper venom. Purified recombinant mouse factor X restored coagulation in human factor X deficient plasma, demonstrating that mouse factor X is able to functionally interact with the human blood coagulation system. Recombinant mouse factor X opens the possibility to analyze therapeutically useful variants in the mouse system.


Subject(s)
Factor X/genetics , Amino Acid Sequence , Animals , Base Sequence , Blood Coagulation/drug effects , Cattle , Cell Line , Cloning, Molecular , Conserved Sequence , DNA, Complementary/genetics , Factor X/isolation & purification , Factor X/pharmacology , Gene Expression , Genetic Variation , Humans , In Vitro Techniques , Mice , Molecular Sequence Data , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Sequence Homology, Amino Acid , Species Specificity
8.
Nat Biotechnol ; 15(7): 629-31, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9219263

ABSTRACT

We describe the engineering of antibody fragments produced in bacteria for recruitment of complement effector functions. From a phage display repertoire we isolated human antibody fragments directed against complement C1q, and linked these to lysozyme-specific antibody fragments, creating bispecific antibodies (diabodies). One diabody was able to recruit C1q, resulting in efficient lysis of lysozyme-coated sheep erythrocytes, and also induced rosette-formation of erythrocytes with human monocytes and phagocytosis after phorbol ester stimulation. These diabodies may have therapeutic applications requiring the activation of complement.


Subject(s)
Antibodies, Bispecific/pharmacology , Complement System Proteins/metabolism , Animals , Antibodies, Bispecific/genetics , Antibodies, Bispecific/metabolism , Base Sequence , Biotechnology , Complement Activation , Complement C1q/metabolism , Erythrocytes/immunology , Hemolysis , Humans , In Vitro Techniques , Monocytes/immunology , Muramidase/immunology , Oligodeoxyribonucleotides/genetics , Protein Engineering
9.
Immunotechnology ; 3(2): 137-44, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9237098

ABSTRACT

BACKGROUND: Bispecific antibodies with a first binding specificity to a target antigen and a second to an enzyme have great potential in enzyme immunoassays. As bispecific antibodies are difficult to make, the use of recombinant bispecific antibody fragments may provide a breakthrough. OBJECTIVES: To make bispecific antibody fragments directed against an enzyme and to demonstrate their application in enzyme immunoassays. STUDY DESIGN: Bispecific antibody fragments were assembled as diabodies (Holliger P., Prospero T., Winter G. Proc. Natl. Acad. Sci. USA 90, 1993, 6444-6448) directed to an enzyme, E. coli beta-galactosidase, and to each of three target antigens, hen-egg lysozyme (HEL), carcinoembryonic antigen (CEA), and HIV gpl20 (HIV). The diabodies were then evaluated in immunoassays. RESULTS: The HEL diabody was shown to recruit beta-galactosidase in a microtiter plate immunoassay in which diabody and enzyme were co-incubated with antigen, washed and enzyme substrate added. The CEA diabody was shown to detect CEA by immunocytochemical staining of transfected, CEA-expressing HeLa cells and of adenocarcinoma colon tissue sections, and the HIV diabody to detect gpl20 in immunoblots of total cell extracts. CONCLUSION: The results illustrate the diagnostic potential of diabodies in enzyme immunoassays.


Subject(s)
Immunoenzyme Techniques , Immunoglobulin Fragments/chemistry , Amino Acid Sequence , Base Sequence , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , Humans , Immunoblotting , Immunoenzyme Techniques/instrumentation , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/isolation & purification , Immunohistochemistry , Molecular Sequence Data , Peptide Library , Sequence Analysis, DNA , beta-Galactosidase/genetics , beta-Galactosidase/immunology
10.
Proc Natl Acad Sci U S A ; 93(15): 7761-6, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755549

ABSTRACT

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase. One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.


Subject(s)
Bacteriophage P1 , Escherichia coli/genetics , Exons , Peptides/chemistry , Peptides/pharmacology , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Enzyme-Linked Immunosorbent Assay , Evolution, Molecular , Glucuronidase/antagonists & inhibitors , Glucuronidase/biosynthesis , Introns , Models, Genetic , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Peptides/chemical synthesis , Polymerase Chain Reaction , RNA, Ribosomal/biosynthesis , RNA, Ribosomal/genetics , Recombinant Fusion Proteins/biosynthesis , Recombination, Genetic , Tetrahymena thermophila/genetics
11.
Biol Chem Hoppe Seyler ; 376(8): 473-81, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7576246

ABSTRACT

The epitope recognized by monoclonal antibody MAb215 generated previously against Drosophila melanogaster RNA polymerase II was mapped to amino acid residues 806-820 of the largest, 215 kDa, subunit located in a region conserved within the largest subunits of pro- and eukaryotic RNA polymerases. The affinities of MAb215 and of a recombinant single-chain Fv fragment (scFv215) were determined for binding to the enzyme as well as the fusion protein and synthetic peptides used for epitope mapping. In addition, amino acid residues of the epitope important for binding to MAb215 were identified using peptides carrying single amino acid substitutions. The epitope is not involved in the polymerization reaction or the DNA unwinding process since no inhibitory effects of the monoclonal antibody were observed in nonspecific in vitro transcription using denatured calf thymus DNA or double stranded oligo dC-tailed T7 DNA as template. In contrast, MAb215 inhibits accurate in vitro transcription from the Krüppel gene promoter and from the adenovirus-2 major late promoter. Preincubation of template DNA with the nuclear extract had no effects on inhibition supporting the notion that the epitope does not participate directly in the formation of preinitiation complexes. The same inhibitory effects were observed using scFv215. The results provide further evidence that recombinant antibody fragments produced in Escherichia coli possess the same specificity and similar affinity as their parental antibodies and demonstrate that scFv fragments are useful tools for analysis of transcriptional processes.


Subject(s)
Drosophila/metabolism , Epitopes/analysis , RNA Polymerase II/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Antigen-Antibody Reactions , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Epitope Mapping , Immunoblotting , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/immunology , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/metabolism , Serum Albumin, Bovine , Transcription, Genetic
12.
Cell Biophys ; 26(3): 187-204, 1995 Jun.
Article in English | MEDLINE | ID: mdl-8521451

ABSTRACT

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.


Subject(s)
Escherichia coli/genetics , Immunoglobulin Variable Region/immunology , Amino Acid Sequence , Animals , Cysteine/chemistry , Escherichia coli/immunology , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Mice , Molecular Sequence Data , Plasmids , Protein Folding , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology
13.
Mol Immunol ; 31(14): 1047-58, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7935496

ABSTRACT

A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli. Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster. ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E. coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents. In a final step, the components were separated by size exclusion chromatography. All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.


Subject(s)
Antibodies, Bispecific/biosynthesis , Antibodies, Bispecific/chemistry , Biotin/chemistry , Immunoglobulin Fragments/chemistry , Immunoglobulin Variable Region/chemistry , Amino Acid Sequence , Animals , Antibody Affinity , Base Sequence , Blotting, Western , Chromatography, Affinity , Cysteine/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Mice , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry
14.
EMBO J ; 13(14): 3245-60, 1994 Jul 15.
Article in English | MEDLINE | ID: mdl-8045255

ABSTRACT

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.


Subject(s)
Antibody Affinity/genetics , Gene Library , Genes, Immunoglobulin/genetics , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Antibody Specificity , Bacteriophage P1/genetics , Base Sequence , Escherichia coli/genetics , Gene Rearrangement , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/genetics , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Selection, Genetic
16.
FEBS Lett ; 344(2-3): 166-70, 1994 May 16.
Article in English | MEDLINE | ID: mdl-8187877

ABSTRACT

Analysing overlapping bacterially expressed fragments of the second-largest subunit of Drosophila melanogaster RNA polymerase II in Southwestern DNA binding assays we have identified regions that have the potential to bind nucleic acids non-specifically. A region exhibiting strong DNA binding is located in the N-terminal part of the molecule (amino acids 357-504) and some weak DNA binding is observed for the C-terminal part (amino acids 860-1160). The non-specific DNA binding behavior of these regions is similar to that of the native enzyme. Most of the known mutations responsible for rifampicin resistance map to a region of the Escherichia coli beta subunit corresponding to the N-terminal nucleic acid-binding region, indirectly supporting the notion that this region participates in interaction with the RNA transcript in ternary complexes.


Subject(s)
DNA/metabolism , Drosophila melanogaster/enzymology , RNA Polymerase II/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Blotting, Southern , Blotting, Western , Isoelectric Point , Molecular Sequence Data , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , beta-Galactosidase/genetics
17.
FEBS Lett ; 335(1): 73-5, 1993 Nov 29.
Article in English | MEDLINE | ID: mdl-8243669

ABSTRACT

The RPII15 gene product of Drosophila melanogaster, which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II. Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies. Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence. Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster. This analysis identified the M(r) 15,000 subunit 9 as gene product of RPII15.


Subject(s)
Drosophila melanogaster/genetics , RNA Polymerase II/genetics , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/genetics , Drosophila melanogaster/enzymology , Electrophoresis, Polyacrylamide Gel , Gene Expression , Immunoblotting , Maltose-Binding Proteins , Molecular Sequence Data , Polymerase Chain Reaction , RNA Polymerase II/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
18.
Protein Sci ; 2(2): 223-30, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8443600

ABSTRACT

The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila melanogaster/enzymology , Nucleic Acids/metabolism , RNA Polymerase II/metabolism , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA Mutational Analysis , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Heparin/pharmacology , Macromolecular Substances , Magnesium Chloride/pharmacology , Molecular Sequence Data , Nucleic Acids/drug effects , Nucleic Acids/genetics , Polymerase Chain Reaction , RNA Polymerase II/drug effects , RNA Polymerase II/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Sodium Chloride/pharmacology , Structure-Activity Relationship
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