RESUMO
We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0.5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity.
Assuntos
Oxirredutases do Álcool/genética , Glutamato Descarboxilase/biossíntese , Pichia/genética , Oxirredutases do Álcool/biossíntese , Oxirredutases do Álcool/química , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Clonagem Molecular , Endopeptidases/metabolismo , Fermentação , Expressão Gênica , Genes Fúngicos , Glutamato Descarboxilase/química , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/isolamento & purificação , Humanos , Metanol/metabolismo , Dados de Sequência Molecular , Mutagênese , Pichia/enzimologia , Pichia/crescimento & desenvolvimento , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Transformação Genética , VacúolosRESUMO
The molecular cloning of mouse plasma phospholipid transfer protein (PLTP) and the eukaryotic cell expression of complementary DNA for mouse and human PLTP are described. Mouse PLTP was found to share 83% amino acid sequence identity with human PLTP. PLTP was produced in baby hamster kidney cells. Conditioned medium from BHK cells expressing PLTP possessed both phospholipid transfer activity and high density lipoprotein (HDL) conversion activity. PLTP mRNA was detected in all 16 human tissues examined by Northern blot analysis with ovary, thymus, and placenta having the highest levels. PLTP mRNA was also examined in eight mouse tissues with the highest PLTP mRNA levels found in the lung, brain, and heart. The effect of purified human plasma-derived PLTP and human recombinant PLTP (rPLTP) on the two human plasma HDL subspecies Lp(A-I) and Lp(A-I/A-II) was evaluated. Plasma PLTP or rPLTP converted the two distinct size subspecies of Lp(A-I) into a larger species, an intermediate species, and a smaller species. Lp(A-I/A-II) particles containing multiple size subspecies were significantly altered by incubation with either plasma or rPLTP with the largest but less prominent subspecies becoming the predominant one, and the smallest subspecies increasing in concentration. Thus, PLTP promoted the conversion of both Lp(A-I) and Lp(A-I/A-II) to populations of larger and smaller particles. Also, both human PLTP and mouse rPLTP were able to convert human or mouse HDL into larger and smaller particles. These observations suggest that PLTP may play a key role in extracellular phospholipid transport and modulation of HDL particles.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Expressão Gênica , Lipoproteínas HDL/sangue , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Proteínas de Transferência de Fosfolipídeos , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Proteínas de Transporte/química , Linhagem Celular , Cricetinae , Meios de Cultivo Condicionados , DNA Complementar/química , Humanos , Rim , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Proteínas Recombinantes/farmacologia , Homologia de Sequência , Distribuição TecidualRESUMO
Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.
Assuntos
Proteínas de Neoplasias , Receptores de Citocinas , Trombopoetina/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 3 , Primers do DNA/química , DNA Complementar/genética , Expressão Gênica , Genes , Humanos , Hibridização in Situ Fluorescente , Megacariócitos/citologia , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Receptores Imunológicos/metabolismo , Receptores de Trombopoetina , Proteínas Recombinantes , Trombopoetina/farmacologiaRESUMO
Phospholipid transfer protein, with an apparent molecular mass of 81 kDa, was purified from human plasma. The NH2-terminal amino acid sequence of a 51-kDa proteolytic fragment obtained from phospholipid transfer protein allowed degenerate primers to be designed for polymerase chain reaction and the eventual isolation of a full-length cDNA from a human endothelial cDNA library. The cDNA is 1,750 base pairs in length and contains an open reading frame of 1,518 nucleotides encoding a leader of 17 amino acids and a mature protein of 476 residues. Northern blot analysis shows a single mRNA transcript of approximately 1.8 kilobases with a wide tissue distribution. The gene was mapped to chromosome 20 using a human/rodent somatic cell hybrid mapping panel. Phospholipid transfer protein was found to be homologous to human cholesteryl ester transfer protein, human lipopolysaccharide-binding protein, and human neutrophil bactericidal permeability increasing protein (20, 24, and 26% identity, respectively).
