RESUMO
Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor-proNGF in vivo. In this paper, a pET-proNGF prokaryocyte expression vector was constructed and transformed into E. coli BL21(DE3)pLysS. The proNGF was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was successfully purified by Ni2+-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment. The protein was dialyzed in a redox system containing reduced and oxidized glutathione. RP-HPLC was used to analysis the result of the refolding. The refolded proNGF protein can induce neurite outgrowth of PC12 cells, which indicated that pro-form of NGF we obtained had biological activity.
Assuntos
Escherichia coli/metabolismo , Fator de Crescimento Neural/biossíntese , Precursores de Proteínas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Fator de Crescimento Neural/genética , Precursores de Proteínas/genética , Renaturação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
AIM: To clone human liver special F protein and to express it in a prokaryotic system. METHODS: Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E. coli BL21 (DE3) pLyss. Isopropy-beta-D-thiogalactoside (IPTG) was then used to induce expression of the target protein. RESULTS: The cDNA clone of human liver special F protein (1134bp) was successfully produced, with the cDNA sequence being published in Gene-bank: DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION: F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.
Assuntos
Clonagem Molecular/métodos , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas/metabolismo , DNA Complementar/genética , Escherichia coli , Vetores Genéticos , Humanos , Fígado/citologia , Proteínas/genética , RNA Mensageiro/genéticaRESUMO
The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex. In this report, the effect of 5'-UTR in the expression of hGH gene in cultured Sf9 cells was examined. A 18 bp length in the end of 5'-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR. The truncated hGH cDNA, delta 1hGH was cloned in pFastBac1, named pFast-Bac-delta 1hGH. After transforming into E. coli. DH10Bac, which have a shuttle vetor-Bacmid, the delta 1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-delta 1hGH DNA was acquired. By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-delta 1hGH was obtained, and hGH gene was expressed. Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the amount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-delta 1hGH containing the truncated 5'UTR was four to five times higher than that infected with rAcV-Bac-hGH.
Assuntos
Regiões 5' não Traduzidas/genética , Hormônio do Crescimento Humano/genética , Insetos/genética , Animais , Sequência de Bases , Células Cultivadas , DNA Complementar/genética , Regulação da Expressão Gênica , Vetores Genéticos/genética , Hormônio do Crescimento Humano/metabolismo , Humanos , Immunoblotting , Insetos/citologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
One of the prominent cell cycle-related modifications of histone proteins whose function is correlated with chromosome condensation is the phosphorylation of histone H3. In this work we used immunofluorescence labeling on human MCF-7 cells with the antibody that was specific for phosphorylated histone H3 at Ser10 to examine the cellular distribution of this protein. The acid-soluble proteins from interphase and mitotic cells were separated by SDS-PAGE and the transferred proteins were probed with the antibody. A strong H3-specific band was only detected in the acid-soluble proteins from mitotic cells, demonstrating the correlation between H3 phosphorylation and mitosis. With confocal microscopy on whole cells, our results showed that mitotic phosphorylation of H3 initiated in discrete foci near the nuclear envelope in early prophase cells. Following initiation, H3 phosphorylation appeared to spread throughout the condensing chromatin and reached maximum in early metaphase cells. Dephosphorylation of H3 began in anaphase cells and was complete immediately prior to detectable chromosome decondensation in telophase cells. There was a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. The possible functions of the singular phosphorylation of the amino-terminus of H3 were discussed.