ABSTRACT
Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (10Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5T and to construct new artificial TNF-binding proteins. We obtained a 10Fn3 gene combinatorial library and screened it using the bacterial display method. After expression of the selected 10Fn3 variants in Escherichia coli cells and analysis of their TNF-binding activity, we identified proteins that display high affinity for TNF and characterized their properties.
Subject(s)
Carrier Proteins/metabolism , Escherichia coli/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Fibronectin Type III Domain , Humans , Plasmids/genetics , Plasmids/metabolism , Protein Engineering , Psychrobacter/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Tumor Necrosis Factors/chemistry , Tumor Necrosis Factors/metabolismABSTRACT
The primary culture of rat cerebellar neurons was used to study protein kinase C activity, intracellular variations in calcium concentration ([Ca(2+)]i), changes in the mitochondrial potential, and neuronal death during hyperstimulation of glutamate receptors and after 24-h incubation with phorbol ester. Prolonged exposure of neurons to glutamate (100 microM, 45 min) was followed by the development of delayed calcium dysregulation. Protein kinase C activity depended on the time of cell incubation with glutamate. Protein kinase C activity increased in response to application of glutamate for 15 min. However, protein kinase C activity decreased after 45-min exposure to glutamate and development of delayed calcium dysregulation. Protein kinase C activity was nearly undetected after 24-h preincubation of neurons with phorbol ester. Under these conditions, delayed calcium dysregulation developed more slowly and was observed in a smaller number of neurons. Neuronal death decreased to 2+/-1%. Our results suggest that protein kinase C plays an important role in death of neurons, which exhibit delayed calcium dysregulation during glutamate treatment.
Subject(s)
Calcium/metabolism , Neurons/metabolism , Protein Kinase C/metabolism , Receptors, Glutamate/metabolism , Animals , Apoptosis , Cells, Cultured , Glutamic Acid/pharmacology , Homeostasis , Membrane Potential, Mitochondrial , Neurons/cytology , Neurons/drug effects , Rats , Receptors, N-Methyl-D-Aspartate/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
In order to design ribozymes for the efficient cleavage of a human acetylcholinesterase (AChE) in vitro transcript, a completely randomized decadeoxyribonucleotide (dN10) was used in conjunction with RNase H to identify suitable sites for annealing. Based on the observed cleavage pattern, ribozymes were designed to cleave the transcript at these positions. Five ribozymes so designed proved to be efficient in the transcript cleavage (k(react)/Km ranged from 0.9 x 10(4) to 68.2 x 10(4) M(-1) min(-10)). The best was 150-fold more active than the best designed on the basis of the MFold program. Thus, the RNase H mapping demonstrated a high predictive power for favorable ribozyme cleavage sites. The digestion pattern with RNase H differed dramatically from that observed with the single-strand-specific mung bean nuclease.
Subject(s)
Acetylcholinesterase/genetics , RNA Precursors/metabolism , RNA, Catalytic/metabolism , RNA, Messenger/metabolism , Humans , Kinetics , Models, Molecular , Nucleic Acid Conformation , RNA, Double-Stranded/metabolism , Ribonuclease H/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Substrate SpecificityABSTRACT
The hammerhead ribozyme is one of the smallest ribozymes known and catalyses the site-specific hydrolysis of a phosphodiester bond. This small ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression. The first part of the review summarizes the sequence requirements of the hammerhead, its three-dimensional structure and the proposed mechanism, in addition to ribozyme specificity and turnover. The second part of the review focuses on the in vivo application of the ribozyme. The processes involved in designing ribozymes for efficient cleavage in vivo are described, together with possible delivery strategies.
Subject(s)
RNA, Catalytic/chemistry , Animals , Animals, Genetically Modified , Base Sequence , Catalysis , Crystallography, X-Ray , Kinetics , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , Structure-Activity RelationshipABSTRACT
Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.
Subject(s)
Genes, Synthetic , Hominidae/genetics , Interleukin-1/biosynthesis , RNA, Messenger/biosynthesis , Sialoglycoproteins/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Enhancer Elements, Genetic , Escherichia coli , Gene Expression , Genes , Genetic Vectors , Humans , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/genetics , Introns , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Chain Initiation, Translational , Plasmids , Polymerase Chain Reaction/methods , Prokaryotic Cells , Protein Biosynthesis , RNA, Messenger/chemistry , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Sialoglycoproteins/geneticsABSTRACT
In a search for new restriction endonucleases type II, among forty bacterial strains of the Bacillus genus two strains producing site-specific endonucleases have been found. Endonucleases BbvAIII and BspFI, isolated from B. brevis BLM B-677 and B. species F, are shown to be true isoschisomers of BspMII (Kpn2I) and Sau3AI, respectively.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Bacteriophage lambda/genetics , Base Sequence , Chromatography, Ion Exchange , DNA, Viral/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Molecular Sequence DataABSTRACT
A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.
Subject(s)
Indicators and Reagents , Oligonucleotides/chemistry , Pyrenes/chemistry , Base Sequence , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Polymerase Chain Reaction , Spectrometry, FluorescenceABSTRACT
An approach to the directed genetic recombination in vitro has been devised, which allows for joining, in a predetermined chemical-enzymatic way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA Splicing by Directed Ligation, SDL). The approach makes use of amplification, by several polymerase chain reactions (PCR), of the chosen DNA segments. The corresponding primers contain recognition sites of the class IIS restriction endonucleases, yielding protruding ends of unique primary structures. The protruding ends of the segments to be joined together are structurally predetermined to make them mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.
Subject(s)
DNA/metabolism , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Exons , Gene Amplification , Humans , Interleukin-1/genetics , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Recombination, GeneticABSTRACT
The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue.
Subject(s)
Anemia, Sickle Cell/genetics , DNA Mutational Analysis/methods , Polymerase Chain Reaction/methods , Alleles , Amino Acid Sequence , Base Sequence , DNA/genetics , DNA Probes , Evaluation Studies as Topic , Globins/genetics , Humans , Molecular Sequence Data , Point MutationABSTRACT
An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.
Subject(s)
DNA Restriction Enzymes/metabolism , DNA, Recombinant , DNA/metabolism , Genes, Synthetic , Interleukin-1/genetics , Base Sequence , Cloning, Molecular , Exons/genetics , Introns/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Restriction MappingABSTRACT
An approach to the directed genetic recombination in vitro mediated by synthetic oligodeoxynucleotides and polymerase chain reaction (PCR) is devised, which allows the joining, in a predetermined chemical-enzymatic way, of a series of DNA segments to give a precisely spliced polynucleotide sequence (Artificial Splicing by Directed Ligation, ASDL). The approach can thus lead to the totally processed eukaryotic genes using genomic DNA, with no mRNA needed. This approach has been used for the synthesis of artificial genes of interleukin-1 alpha and, in combination with PCR on the mRNA-cDNA duplex as template, of interleukin-1 receptor antagonist and their analogues, as well as for the modified genes.
