Adaptation of Mammalian Cells to Growth in Serum-Free Media

Sinacore, Martin S.; Drapeau, Denis; Adamson, Sheena

doi:10.1385/mb:15:3:249

Cited by 99 publications

(59 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For selenomethionine labeling, a selenomethionine-containing medium based on a serum-free formulation (25) was made. Only the inorganic and amino acid components of that formulation were used, and both methionine and sodium selenite were omitted.…”

Section: Methodsmentioning

confidence: 99%

X-ray Crystal Structure of Leukocyte Type Core 2 β1,6-N-Acetylglucosaminyltransferase

Pak

Arnoux²,

Zhou

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Leukocyte type core 2 ␤1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Gal␤1-3[GlcNAc␤1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Gal␤1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Gal␤1-3GalNAc at 2.0 and 2.7 Å resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Gal␤1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside disphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.3 is a cis-medial Golgi resident glycosyltransferase that catalyzes the conversion of the core 1 O-glycan to that of the core 2 structure (1, 2). Core 2 O-glycans have been shown to be key ligands in selectin-mediated lymphocyte homing and leukocyte rolling; lymphocyte L-selectins bind to endothelial cell O-glycans containing 6-sulfo sialyl Lewis x on core 2 and extended core 1 structures, whereas neutrophils expressing sialyl Lewis x on core 2 O-glycans bind to E-and P-selectins on endothelial cells (3-5). C2GnT-L is also of considerable interest in the study of tumor metastasis given that its expression is highly correlated with tumor progression in a number of cancers. It is overexpressed in colorectal, lung, and prostate cancer, and recent work has shown that transfection of C2GnT-L into a prostate cancer cell line leads to increased tumor size in an experimental tumor model (6 -8).C2GnT-L is an inverting N-acetylglucosaminyltransferase belonging to family GT-14 (9). It transfers N-acetylglucosamine (GlcNAc), in ␤1,6 linkage, from UDP-GlcNAc to Gal␤1-3Gal-NAc-O-Ser/Thr (core 1) to generate Gal␤1-3[GlcNAc␤1-6]GalNAc-O-Ser/Thr (core 2). C2GnT-L (leukocyte-type or C2GnT-I) along with C2GnT-M (mucin-type or C2GnT-II) (10, 11) and C2GnT-T (thymus-type or C2GnT-III) (12) are the three ␤1,6 N-acetylglucosaminyltransferases involved in the biosynthesis of core 2 branched O-glycans. The three enzymes differ in tissue distribution, and the mammalian homologues show 40 -50% sequence similarity with each other. Divalent metal ions, which have been shown to be essential for catalytic activity in many glycosyltransf...

show abstract

Section: Methodsmentioning

confidence: 99%

X-ray Crystal Structure of Leukocyte Type Core 2 β1,6-N-Acetylglucosaminyltransferase

Pak

Arnoux²,

Zhou

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Because the nanofiber material is PLGA, which is biocompatible and biodegradable, cells cultured with PLGA nanofibers can be transplanted into patients for therapeutic purposes without the removal of the PLGA nanofibers prior to cell transplantation. HEK293 cells can be adapted to grow in suspension (Sinacore et al 2000). However, the adaptation process is time-consuming and costly.…”

Section: Discussionmentioning

confidence: 99%

Efficient formation of cell spheroids using polymer nanofibers

et al. 2011

View full text Add to dashboard Cite

Spheroid culture has been used for suspension cultures of anchorage-dependent cells. In this study, we developed a new method for the suspension cultures of anchorage-dependent animal cells using polymer nanofibers. Poly(lactic-co-glycolic acid) nanofibers (785 nm in average fiber-diameter, 88 μm in average fiber-length) fabricated by the electrospinning method were added to each suspension culture of human embryonic kidney 293 cells and human dermal fibroblasts. As compared to no addition of nanofibers to the suspension cultures, nanofibers enhanced cell spheroid formation, thereby reducing cell death resulting from a lack of cell adhesion. Efficient formation of spheroids in the presence of polymer nanofibers may be useful for the suspension cultures of anchorage-dependent cells.

show abstract

“…However, the adaptation of the adherent cells to the suspension culture requires a number of special techniques and media formulation, which were not available in the 1980s. Today, there are various commercially available media for adaptation to suspension culture [12,13]. Recently, different types of media have been developed that contain components such as Pluronic F-68, which prevents cell aggregation, for adaptation to suspension culture.…”

Section: Development Of Suspension Culture Systemsmentioning

confidence: 99%

Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems

Park

2010

Korean J. Chem. Eng.

View full text Add to dashboard Cite

The market for protein-drugs has steadily increased due their increased use as alternatives to traditional small molecule drugs. While some therapeutic proteins have been produced in microbial systems, mammalian cell systems such as Chinese hamster ovary (CHO) cells are widely used as the host cell system. To increase the efficiency of producing therapeutic proteins, many researchers have attempted to solve the critical problems that occur in mammalian cell systems. As a result, several serum-free media and advanced culture methods have been developed, and protein productivity has increased considerably through the development of efficient selection methods. However, the prevalence of apoptosis during mammalian cell culture still remains a significant problem. Based on the understanding of apoptotic mechanisms and related proteins, anti-apoptotic engineering has steadily progressed. In this study, we review the strategies that have been developed for high-level production of recombinant proteins in the CHO cell system via a selection of clones, target-gene amplification, optimization of culture systems and an inhibition of apoptosis through genetic modification.

show abstract

Adaptation of Mammalian Cells to Growth in Serum-Free Media

Cited by 99 publications

References 30 publications

X-ray Crystal Structure of Leukocyte Type Core 2 β1,6-N-Acetylglucosaminyltransferase

X-ray Crystal Structure of Leukocyte Type Core 2 β1,6-N-Acetylglucosaminyltransferase

Efficient formation of cell spheroids using polymer nanofibers

Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems

Contact Info

Product

Resources

About