RESUMO
A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.
Assuntos
Engenharia Genética/métodos , Vetores Genéticos , Antígenos de Superfície da Hepatite B/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , DNA Recombinante/imunologia , Plasmídeos , Transcrição GênicaRESUMO
A DNA containing a sequence coding for the human growth hormone releasing factor (hGRF) has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 140 base-pair fragment containing initiation and termination signals for translation and appropriate protruding ends for cloning into a newly constructed plasmid vector (pULB1219). Eleven oligodeoxyribonucleotides, from 14 to 31 bases in length, sharing pairwise stretches of complementary regions of at least 13 bases were prepared by phosphotriester solid-phase synthesis. The DNA sequence was designed to take into account the optimal use of E. coli codons. Oligomers were annealed in one step and assembled by ligation. The DNA fragment of the expected size (140 bp) was recovered and cloned into the pULB1219 vector. The expected sequence was confirmed by DNA sequencing.
Assuntos
Clonagem Molecular , Genes Sintéticos , Genes , Hormônio Liberador de Hormônio do Crescimento/genética , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Humanos , Oligodesoxirribonucleotídeos/síntese química , PlasmídeosRESUMO
A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.