RESUMEN
The rice cell suspension culture system is a good way to produce recombinant human proteins, owing to its high biosafety and low production cost. Human Octamer-binding Transcription Factor 4 (Oct4) is a fundamental transcription factor responsible for maintaining human pluripotent embryonic stem cells. Recombinant Oct4 protein has been used to induce pluripotent stem cells. In this study, recombinant Oct4 proteins are produced via a sugar starvation-inducible αAmy3/RAmy3D promoter-signal peptide-based rice recombinant protein expression system. Oct4 mRNAs accumulate in the transgenic rice suspension cells under sugar starvation. The Oct4 recombinant protein is detected in the transgenic rice suspension cells, and its highest yield is approximately 0.41% of total cellular soluble proteins after one day of sugar starvation. The rice cell-synthesized recombinant human Oct4 protein show DNA-binding activity in vitro, which implies that the protein structure is correct for enabling specific binding to the target DNA motif.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Factor 3 de Transcripción de Unión a Octámeros/aislamiento & purificación , Oryza/citología , Células Cultivadas , Contención de Riesgos Biológicos , Regulación de la Expresión Génica de las Plantas , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , alfa-Amilasas/genéticaRESUMEN
YbeA is a 3-methylpseudoridine methyltransferase from Escherichia coli that forms a stable homodimer in solution. It is one of the deeply trefoil 31 knotted proteins, of which the knot encompasses the C-terminal helix that threads through a long loop. Recent studies on the knotted protein folding pathways using YbeA have suggested that the protein knot remains present under chemically denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for urea-denatured YbeA, which will serve as the basis for further structural characterisations using solution state NMR spectroscopy with paramagnetic spin labeled and partial alignment media.
Asunto(s)
Proteínas de Escherichia coli/química , Metiltransferasas/química , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica/efectos de los fármacos , Urea/farmacología , Escherichia coli/enzimologíaRESUMEN
YibK is a tRNA methyltransferase from Haemophilus influenzae, which forms a stable homodimer in solution and contains a deep trefoil 31 knot encompassing the C-terminal helix that threads through a long loop. It has been a model system for investigating knotted protein folding pathways. Recent data have shown that the polypeptide chain of YibK remains loosely knotted under highly denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for YibK and its variant in the presence of 8 M urea. This work forms the basis for further analysis using NMR techniques such as paramagnetic relaxation enhancement, residual dipolar couplings and spin-relaxation dynamics analysis.
