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1.
Zhonghua Fu Chan Ke Za Zhi ; 41(8): 549-53, 2006 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-17083842

RESUMO

OBJECTIVE: To investigate whether the human endometrial carcinoma cells, RL95-2 infected with recombinant Ad-PTEN can steadily produce PTEN protein and enter apoptosis. METHODS: The recombinant adenovirus containing PTEN cDNA was constructed using the method of homologous recombination in bacteria. The viral titer was examined by plaque assay and the expression of PTEN protein was detected by western blot assay. The apoptosis of RL95-2 cells was evaluated as following: flipping of membrane phosphatidylserine (PS) and identification of activating caspase-3 positive cells was determined by flow cytometer (FCM), and furthermore genomic DNA fragmentation was detected by agarose electrophoresis. RESULTS: The recombinant adenovirus encoding PTEN cDNA was successfully constructed, and viral titers of Ad-PTEN were 5 x 10(9) pfu/ml. After infected by Ad-PTEN, the expression of PTEN protein was steady in human RL95-2 cells. After infected by Ad-PTEN for 24, 48, 72 and 96 h, the relative cell number of membrane PS flipping were (6.09 +/- 1.01)%, (9.98 +/- 2.17)%, (11.74 +/- 2.65)%, (27.69 +/- 8.67)%, which significantly increased than control group (P < 0.05), the relative cell number of activated caspase-3 positive were (2.6 +/- 0.5)%, (18.0 +/- 4.4)%, (21.8 +/- 5.1)%, (33.7 +/- 9.9)%, respectively, which significantly increased than control group (P < 0.05), and genomic DNA fragmentation was verified also. CONCLUSIONS: The recombinant Ad-PTEN vector is constructed successfully and the expression of specific PTEN is steady in RL95-2 cell line. The expression of PTEN induces RL95-2 cells to apoptosis. PTEN gene may be a novel therapeutic target in endometrial carcinoma.


Assuntos
Adenoviridae/genética , Apoptose/genética , PTEN Fosfo-Hidrolase/genética , Apoptose/fisiologia , Western Blotting , Caspase 3/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA , DNA Complementar/genética , Eletroforese em Gel de Ágar , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Citometria de Fluxo , Vetores Genéticos , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Fatores de Tempo , Transfecção
2.
Protein Expr Purif ; 36(2): 292-9, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15249052

RESUMO

Human glucagon-like peptide-1 (hGLP-1) (7-36) amide, a gastrointestinal hormone with a pharmaceutical potential in treating type 2 diabetes mellitus, is composed of 30 amino acid residues as a mature protein. We report here the development of a method for high-level expression and purification of recombinant hGLP-1 (7-36) amide (rhGLP-1) through glutathione S-transferase (GST) fusion expression system. The cDNA of hGLP-1-Leu, the 31st-residue leucine-extended precursor peptide, was prepared by annealing and ligating of artificially synthetic oligonucleotide fragments, inserted into pBluescript SK (+/-) plasmid, and then cloned into pGEX-4T-3 GST fusion vector. The fusion protein GST-hGLP-1-Leu, expressed in Escherichia coli strain BL21 (DE3), was purified by affinity chromatography after high-level culture and sonication of bacteria. Following cleavage of GST-hGLP-1-Leu by cyanogen bromide, the recombinant hGLP-1-Leu was released from fusion protein, and purified using QAE Sepharose ion exchange and RP C(18) chromatography. After purification, the precursor hGLP-1-Leu was transacylated by carboxypeptidase Y, Arg-NH(2) as a nucleophile, to produce rhGLP-1. Electrospray ionization mass spectrometry showed the molecular weight was as expected. The biological activity of rhGLP-1 in a rat model demonstrated that plasma glucose concentrations were significantly lower and insulin concentrations higher after intraperitoneal injection of rhGLP-1 together with glucose compared with glucose alone (P < 0.001).


Assuntos
Arginina/química , Catepsina A/química , Brometo de Cianogênio/química , Glucagon/química , Glucagon/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Precursores de Proteínas/química , Precursores de Proteínas/isolamento & purificação , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Diabetes Mellitus Tipo 2/tratamento farmacológico , Escherichia coli/química , Escherichia coli/genética , Feminino , Expressão Gênica , Glucagon/administração & dosagem , Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon , Glucose/administração & dosagem , Glucose/análise , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , Insulina/sangue , Leucina/química , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
3.
Biochem J ; 370(Pt 2): 567-78, 2003 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-12457462

RESUMO

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Metaloendopeptidases/genética , Mutação , Sinais Direcionadores de Proteínas/genética , Baculoviridae , Proteínas Ligadas por GPI , Vetores Genéticos , Ácido Glutâmico/metabolismo , Temperatura Alta , Humanos , Proteínas de Membrana , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/metabolismo , Desnaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Protein Pept Lett ; 9(6): 553-6, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12553864

RESUMO

Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A. Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).


Assuntos
Substâncias de Crescimento/química , Substâncias de Crescimento/isolamento & purificação , Proteínas , Cristalização , Cristalografia por Raios X , Escherichia coli , Temperatura Alta , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
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