RESUMO
To produce membrane-permeable human epidermal growth factor (hEGF), a pPTD-hEGF prokaryocyte expression vector was constructed and transformed into E. coli BL 21 (DE3). The PTD-hEGF fusion protein was induced by 0.3 mmol/L of IPTG expressed in the form of inclusion body with an yield of 40% of the total protein in the cells, and then purified by Ni2+ -NTA affinity chromatography. The SDS-PAGE analysis showed a single fusion protein band with a molecular weight of 16 kD. The amino acid sequence was checked by MALDI-TOF-MS analysis. The genetic engineering PTD-hEGF fusion protein obviously promoted the proliferation and growth of the HEK-293 cells in vitro.
Assuntos
Fator de Crescimento Epidérmico/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/biossíntese , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Transdução GenéticaRESUMO
The recombinant human liver prolidase (rh-prolidase, EC 3.4.13.9) from the lysate supernatant of engineering yeast Saccharomyces cerevisiae was purified in two steps employing anion-exchange gradient chromatography (DEAE-Sepharose fast flow) and gel filtration chromatography (Sephacryl S-200 high resolution). The purified recombinant protein furnished a single band with a molecular weight of 56 kD. Intensity scanning of the SDS-PAGE gel revealed that the prolidase accounted for more than 90% of total protein. The optimum pH of the catalytic reaction was 8.0. The enzyme was stimulated by Mn2+, but strongly inhibited by Cu2+ and Zn2+. The rh-prolidase expressed in S. cerevisiae had both dipeptidase and organophosphorus acid anhydrolase activity. It catalyzed the hydrolysis of soman and the dipeptide Gly -Pro. In a detoxification test in vitro, purified rh-prolidase was remarkably efficient at eliminating the toxicity of a lethal dose of soman, with the result that mice survived injection of such a dose.
Assuntos
Dipeptidases/biossíntese , Fígado/enzimologia , Saccharomyces cerevisiae/enzimologia , Clonagem Molecular , Dipeptidases/química , Dipeptidases/isolamento & purificação , Humanos , Proteínas Recombinantes , Proteínas de Saccharomyces cerevisiaeRESUMO
By using the matured embryos of Japonica rice variety Zhonghua No. 11 as explants, rice transformation was performed by Agrobacterium-mediated co-cultivation method, resulting in 1489 independent transgenic rice plants that carry a T-DNA insertion. Genomic DNA gel-blot and PCR analyses showed that 69.8% of the total lines contain the inserted T-DNA. The flanking sequence of T-DNA in transgenic rice plants was analyzed using Tail-PCR. In addition, we have evaluated 1066 T1 transgenic lines on heading days, plant height and panicles per hill, and found different types of mutants from a number of lines.
