RESUMO
A cDNA encoding human lysozyme was isolated from a human placenta cDNA library. The cDNA was 1.5 kb in size and coded for a signal peptide consisting of 18 amino acids and mature lysozyme. The amino acid sequence of the mature lysozyme, deduced from the nucleotide sequence, was identical with the published sequence. In the 3'-noncoding region of the cDNA, an Alu sequence was found in the reverse orientation. In a protein coding region, the human lysozyme cDNA shows 60.1% and 51.3% similarity with chicken lysozyme and human alpha-lactalbumin cDNAs, respectively. When the cDNA was expressed in Saccharomyces cerevisiae, an active and correctly processed human lysozyme was secreted efficiently into the culture medium.
Assuntos
Clonagem Molecular , DNA/genética , Muramidase/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Feminino , Genes , Humanos , Dados de Sequência Molecular , Muramidase/biossíntese , Placenta/enzimologia , Proteínas Recombinantes/biossínteseRESUMO
Saccharomyces cerevisiae secreted human lysozyme in the medium as an active form when the signal peptides of chicken lysozyme and a chicken lysozyme-Aspergillus awamori glucoamylase hybrid were used, whereas it did not synthesize any human lysozyme protein by using the signal peptide of A. awamori glucoamylase. The secreted lysozyme was easily purified and crystallized. On the other hand, Bacillus subtilis secreted an inactive human lysozyme, which seemed to have incorrect disulfide bonds, with the signal peptide of amylase and its mutants. The free energy changes for the membrane translocation of the signal peptides are related to the secretion of human lysozyme in S. cerevisiae, but not in B. subtilis. These results indicate that differences exist between S. cerevisiae and B. subtilis in the secretion of human lysozyme.
Assuntos
Bacillus subtilis/enzimologia , Muramidase/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Cristalização , DNA Recombinante/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Humanos , Muramidase/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/farmacologiaRESUMO
A high level of expression in yeast of a chemically synthesized human lysozyme (hL) gene was achieved by introducing an A-rich DNA fragment just upstream from the ATG start codon. The synthesized recombinant human lysozyme (r-hL) was insoluble and biologically inactive. It was solubilized with 7 M urea (pH 9) from yeast cells and its lytic activity was efficiently regenerated by oxidative renaturation. This renaturation experiment and Western blotting analysis under reducing and non-reducing conditions indicate that the insoluble form might be caused by the formation of incorrect intra- or intermolecular disulfide bonds. The N-terminal amino acid sequence of the purified r-hL was identical with that of authentic hL.
