RESUMO
The possibility of DNA-binding proteins interacting in vitro with the polymerase II transcriptional machinery was explored by using a competition assay with individual target sequences for enhancer-binding factors. Transcription factors binding to at least five specific enhancer sequences mediate the activity of the human cytomegalovirus immediate-early 1 gene in vitro. Furthermore, our data suggest that individual DNA-bound enhancer factors can interact with the promoter transcription complex.
Assuntos
Citomegalovirus/genética , Elementos Facilitadores Genéticos , Genes Virais , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Cinética , PlasmídeosRESUMO
In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediate-early promoter of human cytomegalovirus exert a differential effect on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IE1 gene promoter-enhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.
Assuntos
Citomegalovirus/genética , Genes Virais , Linhagem Celular , Citomegalovirus/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Elementos Facilitadores Genéticos , Humanos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.
Assuntos
DNA Complementar/biossíntese , Fator VIII/biossíntese , Glândulas Mamárias Animais/metabolismo , Leite/química , Suínos/genética , Animais , Animais Geneticamente Modificados , Coagulação Sanguínea/efeitos dos fármacos , Dimerização , Fator VIII/genética , Fator VIII/farmacologia , Feminino , Regulação da Expressão Gênica/genética , Hemofilia A/tratamento farmacológico , Hemofilia A/economia , Humanos , Camundongos , Proteínas do Leite/genética , Proteínas Recombinantes/biossíntese , TrombinaRESUMO
The promoter sequences from -175 to +10 of the mouse whey acidic protein (WAP) gene are a target for sequence-specific binding of nuclear proteins [(1987) Nucleic Acids Res. 15, 2103-2121]. Using in vitro transcription assays based on nuclear extracts, transcription factors were shown to bind to these sequences and promoter upstream sequences were found to stimulate transcription.
Assuntos
Proteínas do Leite/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Enzimas de Restrição do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Vitamin K-dependent proteins are currently purified from pooled human plasma or produced in mammalian cell culture systems by recombinant DNA technology. Transgenic animals may provide an additional expression system for the production of these therapeutic proteins. Mice have been used to test hybrid genes which direct the expression of recombinant factor IX and Protein C to the mammary gland. Transgenic livestock have also been created that secrete into milk fully active Protein C at 0.3 mg/mL, and factor IX at 0.2 mg/mL. Thus, it is feasible to develop genetically manipulated livestock for the industrial production of vitamin K-dependent proteins.
Assuntos
Proteínas Sanguíneas/biossíntese , Vitamina K/farmacologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Fator IX/biossíntese , Humanos , Proteína C/biossíntese , Proteínas Recombinantes/biossínteseRESUMO
The binding of nuclear proteins and the functional activity of the HIV-LTR enhancer repeats in different cell lines (Jurkat, CEM, H9, U937, Raji, B cells, T47D, HeLa, 293, and HepG2 cells) was investigated in vitro. Five distinct complexes formed with the enhancer repeat have been identified by an electrophoretic mobility shift assay. The distribution of these complexes varied qualitatively and quantitatively between nuclear proteins from different sources. In the extracts tested, transcription of the HIV-LTR 5' deletion mutants (-453/80, -176/80, -117/80, -103/80, -65/80, and -48/80) was initiated correctly. Transcriptional stimulation dependent upon the presence of the enhancer repeat sequences was observed in all nuclear extracts and was highest in Jurkat, Raji, and B cell extracts. The presence of specific factors and the functional activity of the enhancer repeats as well as other regulatory units in a variety of cells indicates limited host-cell restriction of HIV transcription initiation in vitro.
Assuntos
Elementos Facilitadores Genéticos , HIV/genética , Sequências Repetitivas de Ácido Nucleico , Linhagem Celular , Citomegalovirus/genética , DNA Viral/genética , Humanos , Mutação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição GênicaRESUMO
The regulatory elements of genes used to target the tissue-specific expression of heterologous human proteins have been studied in vitro and in transgenic mice. Hybrid genes exhibiting the desired performance have been introduced into large animals. Complex proteins like protein C, factor IX, factor VIII, fibrinogen and hemoglobin, in addition to simpler proteins like alpha 1-antitrypsin, antithrombin III, albumin and tissue plasminogen activator have been produced in transgenic livestock. The amount of functional protein secreted when the transgene is expressed at high levels may be limited by the required posttranslational modifications in host tissues. This can be overcome by engineering the transgenic bioreactor to express the appropriate modifying enzymes. Genetically engineered livestock are thus rapidly becoming a choice for the production of recombinant human blood proteins.
Assuntos
Animais Geneticamente Modificados , Reatores Biológicos , Proteínas Sanguíneas/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Transfusão de Componentes Sanguíneos , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossínteseRESUMO
Maturation of human Protein C (HPC) precursor to a zymogen in the liver requires endoproteolytic cleavages after a basic dipeptide, Lys-2-Arg-1 in the propeptide and Lys156-Arg157 connecting the light and heavy chain. Recombinant human Protein C (rHPC) was expressed in the mammary gland of transgenic swine and its proteolytic processing was monitored. We found that about 10-20% of rHPC purified from the milk still retained the propeptide and 30-40% was in the single-chain form, indicating inefficient proteolytic cleavage. This demonstrates that endoprotease(s) of the swine mammary epithelial cells do not process fully the precursor of heterologous protein. rHPC was fractionated by anion exchange chromatography and polypeptides with novel N-termini at positions -1,152 and 157 were detected in addition to the known N-termini at residues -24, +1, and 158. Since rHPC was found to be stable both in milk and after purification, it is possible that these new cleavages on the amino side of arginine at dipeptide sites Lys-2-Arg-1, Lys151-Arg152, and Lys156-Arg157 could have occurred in the mammary gland. Thus, our results suggest that a portion of HPC precursor was proteolytically processed in swine mammary gland differently than those in other expression systems.
Assuntos
Glândulas Mamárias Animais/metabolismo , Proteína C/genética , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Endopeptidases , Feminino , Humanos , Hidrólise , Dados de Sequência Molecular , Proteína C/biossíntese , Proteínas Recombinantes/biossíntese , SuínosRESUMO
Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.
Assuntos
Glândulas Mamárias Animais/metabolismo , Proteína C/biossíntese , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , DNA Complementar , Feminino , Humanos , Cinética , Lactação , Camundongos , Camundongos Transgênicos , Leite/química , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteína C/genética , Proteína C/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , SuínosRESUMO
The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.
Assuntos
Biotecnologia , Glândulas Mamárias Animais/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Animais Geneticamente Modificados , Feminino , Glicosilação , Humanos , Cinética , Glândulas Mamárias Animais/anatomia & histologia , Leite/química , Estrutura Molecular , Proteína C/biossíntese , Proteína C/química , Proteína C/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Suínos , Vitamina K/metabolismoRESUMO
We have produced recombinant human Protein C (rHPC) in the milk of transgenic swine. After purification, we have analyzed the interaction of teh zymogen with Protac, thrombin/thrombomodulin and thrombin alone. The amidolytic and anticoagulant activities of rAPC after Protac activation were approximately 80% those of its human plasma counterpart. Upon the excision of the activation peptide by thrombin/thrombomodulin complex, both the natural and recombinant activation products had similar enzymatic and biological activities. This observation can be attributed to the difference in the mechanism of action between the two activators and structural differences between HPC and rHPC.
Assuntos
Anticoagulantes/metabolismo , Proteína C/metabolismo , Animais , Animais Geneticamente Modificados , Anticoagulantes/agonistas , Anticoagulantes/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/farmacologia , Proteína C/agonistas , Proteína C/farmacologia , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Suínos , Trombina/química , Trombomodulina/químicaRESUMO
The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.
Assuntos
Cromatina/análise , Proteínas Cromossômicas não Histona/análise , Fígado/análise , Fatores Etários , Animais , Embrião de Galinha , Galinhas , DNA/análise , Histonas/análise , Fígado/ultraestrutura , Fosfoproteínas/análise , RNA/análiseRESUMO
DNA, histones and nonhistone proteins were determined in the chromatin isolated from liver nuclei of the 2-, 9-, 16-, 23-, 30-day-old adult chickens. Very little variation was observed in the total histone/DNA ratio and the electrophoretic mobilities of histones, whereas some differences, mostly of a quantitative character, were found in the nonhistone fraction of chromatin proteins.
Assuntos
Cromatina/metabolismo , Fígado/metabolismo , Envelhecimento , Animais , Núcleo Celular/análise , Fenômenos Químicos , Química , Galinhas , Eletroforese em Gel de Poliacrilamida , Histonas/metabolismo , Fígado/crescimento & desenvolvimento , Peso Molecular , Proteínas/metabolismoRESUMO
Milk of transgenic pigs secreting recombinant human Protein C (rHPC) was used as a model system to determine the utility of aqueous two-phase extraction systems (ATPS) for the initial step in the purification of proteins from milk. The major challenges in purification of recombinant proteins from milk are removal of casein micelles (that foul processing equipment) and elimination of the host milk proteins from the final product. When milk was partitioned in ATPS composed of polyethylene glycol (PEG) and ammonium sulfate (AS), the phases were clarified and most of the caseins precipitated at the interphase. The partition coefficients of the major milk proteins and rHPC were dependent upon the molecular weight of the PEG used in the ATPS. Higher-partition coefficients of the major whey proteins, beta-lactoglobulin, and alpha-lactalbumin were observed in ATPS made up of lower molecular-weight PEG (1000 or 1450) as compared to systems using higher molecular-weight PEG. Lowering the pH of the ATPS from 7.5 to 6.0 resulted in increased precipitation of the caseins and decreased their concentration in both phases. rHPC had a partition coefficient of 0.04 in a system composed of AS and PEG 1450. The rHPC in pig milk was shown to be highly heterogenous by two-dimensional gel electrophoresis. The heterogeneity was owing to inefficient proteolytic processing of the single chain to the heterodimeric form and differences in glycosylation and other post-translational processing. Differential partitioning of the multiple forms of purified rHPC in the ATPS was not observed. rHPC after processing in ATPS was recovered in a clear phase free of most major milk proteins. ATPS are useful as the initial processing step in the purification of recombinant proteins from milk because clarification and enrichment in combined in a single step.
Assuntos
Proteínas do Leite/isolamento & purificação , Leite/química , Proteína C/biossíntese , Proteína C/isolamento & purificação , Sulfato de Amônio , Animais , Animais Geneticamente Modificados , Caseínas/isolamento & purificação , Precipitação Química , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Micelas , Polietilenoglicóis , Proteína C/genética , Especificidade da Espécie , SuínosAssuntos
Proteínas de Ligação a DNA/metabolismo , DNA/genética , Genes , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular/métodos , Células HeLa/metabolismo , Humanos , Linfócitos/metabolismo , Proteínas do Leite/genética , Radioisótopos de Fósforo , Técnica de Diluição de RadioisótoposAssuntos
Animais Geneticamente Modificados/metabolismo , Proteínas Sanguíneas/isolamento & purificação , Proteínas do Leite/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Proteínas Sanguíneas/biossíntese , Proteínas Sanguíneas/economia , Proteínas Sanguíneas/genética , Bovinos , Custos e Análise de Custo , Regulação da Expressão Gênica , Cabras , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/economia , Proteínas Recombinantes de Fusão/genética , Ovinos , Especificidade da Espécie , Suínos , Estados Unidos , United States Food and Drug AdministrationAssuntos
Animais Geneticamente Modificados/metabolismo , Leite/metabolismo , Biossíntese de Proteínas , Animais , Reatores Biológicos , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Proteína C/biossíntese , Proteína C/uso terapêutico , Engenharia de Proteínas , Proteínas Recombinantes , SuínosRESUMO
Nuclear extracts from mammary glands of lactating rats contain DNA binding protein(s) with high affinity to the region -125 to -85 of the rat alpha-lactalbumin promoter. The protected sequence is part of a mammary conserved sequence described by Hall et al. [(1987) Biochem. J. 242, 735-742]. Comparison of the DNA protein-binding sites in the 5' flanking sequence of the rat alpha-lactalbumin and mouse whey acidic protein genes suggests that the consensus sequence 5'-TGGCARNNWCKKC-3' is the protein(s) recognition site. This binding site is part of a highly conserved region [consensus sequence, TGGCAGSCTCGGCST(G)YTCTCTCT(NTG)TGGCARA] present in the promoter of four whey protein genes.
Assuntos
Lactalbumina/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Ligação Competitiva , Proteínas de Ligação a DNA/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Camundongos , Proteínas do Leite/genética , Proteínas Nucleares/metabolismo , Gravidez , Ligação Proteica , Ratos , Proteínas do Soro do LeiteRESUMO
The gene for the whey acidic protein (WAP) is expressed specifically in the lactating mammary glands of rodents. We present evidence that nuclear proteins from mammary epithelial cells form a multiple nucleoprotein complex with the WAP gene promoter/upstream region. As monitored by mobility shifts, nuclear proteins from lactating mammary glands and from the mammary cell line MCF-7 form four high affinity complexes with a fragment spanning the region between nucleotides -175 and -88. Nuclear proteins from liver and HeLa cells generate only three high affinity complexes. DNAaseI and ExonucleaseIII protection confirmed the binding of mammary nuclear proteins to specific sequences in the WAP gene upstream region. This is the first report to describe the interaction of nuclear proteins from lactating mammary glands with cognate binding sites in the promoter/upstream region of a milk protein gene. The possibility of the binding sites being candidates for cis-acting regulatory elements governing the regulated expression of the WAP gene is discussed.