Controlling N-linked glycan site occupancy.

N-linked glycosylation, a common co-translational modification in eukaryotic cells, involves the transfer of a lipid-linked oligosaccharide onto asparagine residues in a tripeptide sequon on a nascent protein in the lumen of the endoplasmic reticulum. The attachment of an oligosaccharide unit to the polypeptide at the site of occupancy can enhance solubility, improve folding, facilitate secretion, modulate antigenicity, and increase in vivo half-life of the glycoprotein. A number of proteins exhibit variable site occupancy.

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells.

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter.

Polyprenyl lipid synthesis in mammalian cells expressing human cis-prenyl transferase.

The level of cis-prenyl transferase activity has been implicated in controlling the level of biosynthesis of dolichol and dolichol intermediates. In this study, we isolated a cDNA encoding a human CPT (GenBank Accession No. ), which had substantial homology to other CPT isolated from human brain, bacteria, Arabidopsis, and Saccharomyces cerevisiae.