RESUMO
A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
Assuntos
Fosfatase Alcalina/genética , Quimosina/genética , Clonagem Molecular , Precursores Enzimáticos/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Animais , Bovinos , Quimosina/biossíntese , Precursores Enzimáticos/biossíntese , Escherichia coli/enzimologia , Plasmídeos , Sinais Direcionadores de Proteínas/biossíntese , Proteínas Recombinantes de Fusão/biossínteseRESUMO
New regulations under the Clinical Laboratory Improvement Act of 1988 have been finalized, but not without objection from the State Society. This article outlines the regulations, compares them to those originally proposed in May 1990, presents a compliance timeline, and touches upon Society concerns.
Assuntos
Centers for Medicare and Medicaid Services, U.S./legislação & jurisprudência , Laboratórios/legislação & jurisprudência , Consultórios Médicos/legislação & jurisprudência , Garantia da Qualidade dos Cuidados de Saúde/legislação & jurisprudência , Humanos , Estados UnidosRESUMO
In an attempt to express the two distal genes of the Escherichia coli threonine operon, the majority of the first gene in the operon, thrA, was removed and a series of transcriptional fusions were constructed placing the thrB and thrC genes downstream of either the trp or hybrid tac promoter. Analysis of the proteins produced by cells containing these fusions revealed that although the distal gene, thrC, was efficiently expressed, the proximal gene, thrB, was not expressed at a detectable level. A translational fusion was constructed which fused the cat gene in phase to the last 800 base pairs of thrA followed by thrB and thrC. Cells containing this fusion produced high levels of both the thrB and thrC gene products, showing that translation of thrB requires translation through thrA; thus, thrA and thrB are translationally coupled. In addition, it was found that a sequence between 220 and 57 base pairs before the start of thrB was necessary to allow translational coupling to occur.