RESUMO
A synthetic gene for human interleukin-6 has been expressed in E. coli. The protein has been purified and renatured and has the same activity as natural human IL-6 using the 7TD1 cell proliferation assay. The protein undergoes specific cleavage by a thiol protease, yielding two new N-termini at Arg-9 and His-15. The truncated proteins retain full biological activity. The degradation results in the loss of sharp amide resonances in the 1H-NMR spectrum, and little change to the ultraviolet CD spectrum. Several amino acid type assignments could be made for these sharp amides using a DQF-COSY 2D-NMR experiment. The N-terminal 15 amino acids exist as a flexible, random coil, attached to a central structure.
Assuntos
Interleucina-6/química , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Escherichia coli/genética , Genes Sintéticos , Humanos , Interleucina-6/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas Recombinantes/químicaAssuntos
Hibridização de Ácido Nucleico/história , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , História do Século XX , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/genética , Microglobulina beta-2/genéticaRESUMO
The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.
Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Interleucina-5/genética , Aminoácidos/análise , Animais , Diferenciação Celular , Fenômenos Químicos , Físico-Química , Dissulfetos/análise , Eletroquímica , Eosinófilos/citologia , Temperatura Alta , Humanos , Recém-Nascido , Interleucina-5/isolamento & purificação , Interleucina-5/farmacologia , Substâncias Macromoleculares , Camundongos , Peso Molecular , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Solubilidade , Compostos de Sulfidrila/análiseRESUMO
Oligodeoxynucleotides have been selectively labeled with biotin at their 5'-termini through an aminoalkylphosphoramide linker arm by an efficient chemical method. The reactions were performed in aqueous solution on unprotected oligonucleotides and were insensitive of the sequence and length of the oligonucleotide. 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters. Detection level is about 2 fmole. There is no noticeable disturbance of the strength and selectivity of hybridization of the 5'-biotin-labeled probes in comparison with non-modified DNA.
Assuntos
Biotina , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos , Oligonucleotídeos , Sequência de Bases , Fenômenos Químicos , Química , DNA Viral/metabolismo , Eletroforese em Gel de Poliacrilamida , Métodos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Plasmídeos , TemperaturaRESUMO
We have synthesized two sets of 15-base-long oligodeoxyribonucleotides corresponding to all possible coding sequences for a small portion of human beta 2-microglobulin. Labeled oligonucleotides were used as hybridization probes to screen bacterial clones containing cDNA sequences primed with oligo(dT) and inserted into the plasmid vector pBR322. One beta 2-microglobulin cDNA clone was detected in the 535 bacterial plasmid clones that were screened. The clone has been characterized by blotting and nucleotide sequence analysis. The cloned beta 2-microglobulin sequence contains 217 base pairs of the 3' untranslated region of the mRNA and 328 base pairs (97%) of the coding region.
Assuntos
beta-Globulinas/genética , Clonagem Molecular , DNA Recombinante/isolamento & purificação , Oligodesoxirribonucleotídeos/síntese química , Oligonucleotídeos/síntese química , Microglobulina beta-2/genética , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Humanos , Hibridização de Ácido Nucleico , PlasmídeosRESUMO
Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.
