RESUMO
The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tet-regulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the trail gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K1 increased, and the proportion of a-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on the biomechanic properties of Jurkat cells.
Assuntos
Membrana Celular/química , Fluidez de Membrana , Glicoproteínas de Membrana/biossíntese , Proteínas Recombinantes/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose , Membrana Celular/metabolismo , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Células Jurkat , Fluidez de Membrana/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Potenciais da Membrana/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Repressoras/genética , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
To express BACE (beta-site APP cleaving enzyme), an aspartic protease related to Alzheimer's disease in E.coli to obtain the active protein after refolding and purification, the sequence for soluble form of BACE was inserted into prokaryotic expression vector pET11a. After expression in E.coli BL21(DE3), the protein was obtained as inclusion bodies, after refolding and purification. The proteolytic activity of the protein was measured by HPLC and MS. After refolding and purification, the protein exhibited beta-secretase activity by cleaving the synthetic peptide, which was designed according to the beta-secretase site at Swedish mutant of APP, and according Hanes method the kinetics constants for the synthetic peptide of BACE protein were measured. The study will facilitate BACE protein structure determination and inhibitor development efforts, which may lead to the evolution of promising and effective treatments for Alzheimer's disease.
