RESUMO
A rapid method for the removal of protecting groups from synthetic oligodeoxyribonucleotides, obtained by the phosphoroamidite and H-phosphonate methods, including the use of hydrazine solutions has been developed. The combination of this procedure with the application of isopropoxyacetyl N-protecting group and oxalyl ester linkage for the first nucleoside attachment to the polymer support allows to reduce to several minutes the time needed for the full oligonucleotide deprotection.
Assuntos
Hidrazinas/química , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Dados de Sequência MolecularRESUMO
The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.
Assuntos
Albuminas/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Streptococcus/metabolismo , Albuminas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , DNA , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Plasmídeos , Sequências Repetitivas de Ácido NucleicoRESUMO
Two new rapid procedures for the full deprotection of synthetic oligonucleotides has been developed. We have successfully used the mixture of ethanolamine and ethanol (1:1) or pure ethanolamine for deprotection of oligonucleotides, prepared by different methods. In the case of oligonucleotides prepared by commonly used beta-cyanoethyl phosphoramidite and H-phosphonates method deprotection takes half an hour at 70 degrees C. We have found also that mixture of hydrazine, ethanolamine and methanol (1:3:3, v/v/v) can serve as a very efficient reagent for deprotection of oligonucleotides, prepared by beta-cyanoethyl phosphoramidite method with isopropoxyacetyl protecting group for cytosine residues. In this case deprotection time is 12-17 min at room temperature.
Assuntos
Oligonucleotídeos/síntese química , Química/métodos , Indicadores e Reagentes , Organofosfonatos , FosfatosRESUMO
Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier. The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized. The constructions obtained were cloned in plasmid vectors.
Assuntos
Genes Sintéticos , Insulina/genética , Sequência de Aminoácidos , Escherichia coli/genética , Engenharia Genética , Humanos , Dados de Sequência Molecular , Plasmídeos , Proinsulina/genética , Precursores de Proteínas/genéticaRESUMO
Expression of the synthetic gene for human proinsulin in E. coli has been investigated. The proinsulin gene has been expressed directly under the control of a synthetic promoter of phage fd DNA and a promoter of tryptophan operon, or using fusions with fragments of some bacterial proteins. These fusions gave insoluble polypeptide products amounting to 20-30% of total cellular protein. The scheme for isolating proinsulin from bacterial cells was developed. Proinsulin was cleaved from leader polypeptides by treatment with cyanogen bromide and converted into human insulin.
Assuntos
Proinsulina/biossíntese , Bacteriófagos/genética , Cromatografia em Gel , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Plasmídeos , Proinsulina/genética , Regiões Promotoras Genéticas , Radioimunoensaio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The chemical-enzymatic synthesis of a gene for IgG-binding fragment of the staphylococcal protein A has been carried out. The design of the gene, which consists of signal peptide and modified E and B domains, and strategy of the synthesis provided possibility of various degrees of polymerization of the gene fragment coding for B domain and of the whole gene. Several protein A-like polypeptides composed of the leader sequence, E domain and 1 to 4 copies of B domain were produced in E. coli cells under the lac promoter control.
Assuntos
Regulação da Expressão Gênica , Genes Sintéticos , Fragmentos de Imunoglobulinas/genética , Proteína Estafilocócica A/genética , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Dados de Sequência Molecular , Staphylococcus aureus/imunologiaRESUMO
Plasmid-based promoter-probe vectors pPV4 and pPV5 have been constructed which are useful for comparing the relative efficiencies of bacterial promoters. The vectors utilize the beta-galactosidase (lacZ) gene of E. coli as an indicator gene. The latter was modified using synthetic DNA fragments. The promotor-probe system contains the ampicillin resistance gene and the origin of replication of plasmid pBR322. The plasmids pPV4 and pPV5 carry clustered unique restriction sites usable for promoter insertions, and SD sequence. A synthetic DNA fragment corresponding to transcription terminator was inserted downstream the lacZ gene. Presence of the terminator made it possible to clone strong promoters controlling transcription of the lacZ gene. To prevent any undesired promotor effect, the plasmid pPV5 has also second synthetic terminator upstream from the polylinker sequence. Using this promoter-probe system, relative efficiencies of a series of synthetic promoters, including PL promoter of phage lambda and its mutant, gene X promotor of phage fd and several model statistic promoters, have been compared.
Assuntos
Escherichia coli/genética , Galactosidases/genética , Genes Bacterianos , Vetores Genéticos , Regiões Promotoras Genéticas , beta-Galactosidase/genética , Resistência a Ampicilina/genética , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , Dados de Sequência Molecular , Plasmídeos , Transcrição GênicaRESUMO
To investigate structure-function relationships in zein proteins and to develop an approach to the construction of mutant zeins of improved nutritional qualities, the chemical-enzymatic synthesis of a gene for zein cZ22B1 (22 kd) has been undertaken. This 806-base pair long DNA fragment consists of about 40 synthetic oligonucleotides, mostly 30-60-mers. The synthesis was planned with the use of a universal methodology for the artificial gene construction. The choice of appropriate sites for altering amino acid sequence and the possibility of obtaining by directed mutagenesis of the gene corresponding to modified zeins containing residues Trp and Lys in specified positions of the polypeptide chain is discussed.
Assuntos
Clonagem Molecular , Genes Sintéticos , Zea mays/genética , Zeína/genética , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Dados de Sequência MolecularRESUMO
The sequence of a stretch of the barley chloroplast DNA containing psbA gene with flanking regions and the deduced amino acid sequence of the photosystem II QB protein have been determined. Comparison of the psbA and QB sequences from barley and some dicotyledonous species shows them to be highly conserved. The sequences of the regions which flank the probable transcription initiation site for psbA gene of barley are identical to the corresponding sequences of wheat.
