RESUMO
AIM: To clone and express soluble B lymphocyte stimulator (sBLyS). METHODS: Total RNA was isolated from peripheral blood mononuclear cells, and used to synthesize cDNA by reverse transcription. sBLyS cDNA was amplified by PCR with specific primers and inserted into a prokaryotic expression vector pET-30a. Recombinant plasmid was transformed into E.coli strain BL21(DE3). sBLyS was expressed in E.coli, purified in vitro, and analyzed with peptide mass fingerprinting and Daudi cell proliferation assay. RESULTS: sBLyS cDNA was cloned. Peptide mass fingerprinting of purified BLyS matched with that of BLyS proteins. Purified sBLyS could stimulate Daudi cell proliferation in vitro. CONCLUSION: sBLyS with biological activity was successfully expressed and purified.
Assuntos
Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Expressão Gênica , Leucócitos Mononucleares/química , Animais , Clonagem Molecular , DNA Complementar/análise , Amplificação de Genes , Vetores Genéticos/genética , Humanos , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da PolimeraseRESUMO
AIM: To prepare recombinant ricin A-chain(RTA) protein with high biological activity. METHODS: RTA gene containing KDEL sequence at the carboxyl terminal was cloned in pET32a vector, which was fused with thioredoxin. Furthermore, the constructed recombinant plasmid was transformed into the competent cell BL21, and induced with low concentration of IPTG (0.4 mmol/L) under low temperature (20 degrees Celsius). After binding to Co2+ chelating column, the expressed supernatants were eluted by applying imidazole solutions with the concentration from 20 to 100 mmol/L. The purified protein was identified with SDS-PAGE and Western blot analysis and was used to cleave supercoiled dsDNA. RESULTS: About 60 mg fusion proteins were obtained from 1,000 mL cultures , with purity above 90% and M(r) 45,000. The cleavage of supercoiled dsDNA demonstrated that RTA-Trx fusion proteins could significantly cleave supercoiled dsDNA as native RTA. CONCLUSION: The pET32a vector expression system can be used to produce a mass of soluble RTA-Trx fusion proteins with high biological activity.
Assuntos
Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Ricina/biossíntese , Ricina/isolamento & purificação , DNA Super-Helicoidal/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ricina/genética , Ricina/metabolismoRESUMO
CapLC-ESI-MS/MS and nano-ESI-MS/MS techniques were used to identify the apoptosis associated proteins induced by inhibiting the ubiquitin-proteasome pathway in Mo7e leukaemic cells. In 2-DE, spot H was found to initiate its overexpression at 2 h after the inhibition and reached its peak at 6 h. It was identified as Rho GDI beta protein after the tandem mass spectrum and after the sequence of its tryptic peptides were obtained by the ESI-MS/MS techniques. It was not revealed by peptide mass fingerprint using MALDI-TOF-MS. Other two spots induced by the inhibition appeared close to spot H were also revealed identical to Rho GDI brg;, possibly due to unknown modifications.
