Video Experimental Relacionado
Updated: Jul 10, 2026
11:25
Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
La glicosilación ligada a N de proteínas plegadas por la oligosacáriltransferasa bacteriana
Michael Kowarik1, Shin Numao, Mario F Feldman
1Institute of Microbiology, Department of Biology, Eidgenössische Technische Hochschule (ETH) Zurich, 8093 Zurich, Switzerland.
Science (New York, N.Y.)
|November 18, 2006
Resumen
La glicosilación bacteriana de proteínas ligadas a N ocurre independientemente de la translocación de proteínas. A diferencia de los eucariotas, los sistemas bacterianos glicosilan las proteínas plegadas de manera eficiente, lo que sugiere distintas vías evolutivas para esta modificación esencial de la proteína.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Molecular Biología Molecular
- Microbiología Microbiología.
Sus antecedentes:
- La glicosilación de proteínas ligadas a N es una modificación post-traducional crucial en todos los dominios de la vida.
- En los eucariotas, es abundante para las proteínas secretoras y de membrana, junto con la translocación y el plegamiento.
- El mecanismo bacteriano y su relación con el plegamiento de las proteínas siguen siendo menos comprendidos.
Objetivo del estudio:
- Para investigar el mecanismo de la glicosilación de proteínas ligadas a N en bacterias.
- Para comparar la glicosilación bacteriana con el proceso cotranslacional eucariótico.
- Para determinar si la glicosilación bacteriana es dependiente de la maquinaria de translocación de proteínas.
Principales métodos:
- Ensayos de glucosilación in vitro con el uso de oligosacáriltransferasa bacteriana.
- Prueba de la eficiencia de la glicosilación en proteínas de sustrato endógenas y exógenas plegadas y desplegadas.
- Análisis de la conformación de la proteína del sustrato (doblada vs. desplegada) en relación con la glicosilación.
Principales resultados:
- La glicosilación ligada a N bacteriana puede ocurrir independientemente de la maquinaria de translocación de proteínas.
- La oligosacáriltransferasa bacteriana glicosilizó eficientemente un sustrato endógeno plegado.
- La glicosilación de la ribonucleasa A bovina plegada fue ineficiente, pero aumentó significativamente al desplegarse.
Conclusiones:
- La N-glicosilación bacteriana difiere del mecanismo cotranslacional de los eucariotas.
- Los sistemas bacterianos pueden glicosilizar sitios flexibles en proteínas ya plegadas.
- La glicosilación cotranslacional eucariota evolucionó para garantizar la flexibilidad del sustrato antes de doblarse para una modificación eficiente.
Videos de Conceptos Relacionados
02:15Protein and Protein Structure
Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective. They may serve in transport, storage, or membranes; or they may be toxins or enzymes. Their structures, like their functions, vary greatly. They are all, however, amino acid polymers arranged in a linear sequence.
A protein's shape is critical to its function. For example, an enzyme can...
A protein's shape is critical to its function. For example, an enzyme can...
01:25Protein Glycosylation
Glycosylation, the most common post-translational modification for proteins, serves diverse functions. Adding sugars to proteins makes the proteins more resistant to proteolytic digestion. Glycosylated proteins can act as markers and receptors to promote cell-cell adhesion. Additionally, they have many essential quality control functions in the cell, such as correct protein folding and facilitating transport of misfolded proteins to the cytosol, which can be degraded.
Glycosylation occurs in...
Glycosylation occurs in...
01:24Oligosaccharide Assembly
Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Multiple sugar molecules that may or may...
01:26Protein Modifications in the RER
Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
01:29Protein Folding Quality Check in the RER
ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm...
01:30Production of Pharmaceuticals
Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...