Skip to content
Login Register

#protein production

4 public questions tagged with this topic.

Animal Tissue Culture 3

CHO cells are extensively used for:

Logarithmic or exponential phase follows lag and is characterized by maximal and constant specific growth rate where population doubles at regular intervals, producing straight line on semi-logarithmic plot of cell number versus time. Birth rate greatly exceeds death rate. Cellular activities peak including DNA replication by DNA polymerases alpha and delta, rRNA transcription, protein synthesis, high glucose consumption via aerobic glycolysis producing lactate, and oxygen uptake by mitochondria. For most continuous lines generation time 18-24 hours though HeLa approximately 20 hours. Contact inhibition not yet engaged, space and growth factors remain sufficient. This phase represents healthiest period with viability above 95% and high fraction of cells in S/G2/M measurable by BrdU or propidium iodide staining. Optimal window for transfection because nuclear envelope breakdown during mitosis enhances plasmid entry, for viral infection requiring active replication machinery, and for cell cycle studies. Maintaining cultures through regular subculture ensures they remain in log phase, avoiding stress responses associated with plateau and death phases that reduce reproducibility.

Ref: Freshney Ch.13 Log phase exponential growth; Lodish MBoC Ch.12 Cell cycle exponential proliferation and growth curve.

View Details
Gene Therapy

Factor VIII recombinant protein is commonly produced in:

Coagulation factor VIII complex multidomain glycoprotein organized signal peptide 19 residues followed domains A1 a1 A2 a2 B a3 A3 C1 C2 totaling 2351 aa 280 kDa requiring extensive co-translational post-translational processing secretory pathway. Nascent polypeptide translocates Sec61 translocon ER lumen signal peptidase cleaves signal oligosaccharyltransferase adds high-mannose oligosaccharides 25 asparagine X Ser Thr motifs protein disulfide isomerase forms 8 disulfide bonds calnexin calreticulin cycle glucosidase II monitors folding peptidyl prolyl isomerase assists. Golgi further trims mannose adds complex sialylated glycans glycosyltransferases sulfates tyrosines 346 718 719 tyrosylprotein sulfotransferase essential vWF binding incorporates copper ions. E. coli cytoplasm lacks ER glycosylation sulfation chaperone BiP leading misfolded aggregates inclusion bodies lacking cofactor activity eliciting neutralizing antibodies exposed neoepitopes. Chinese hamster ovary cells possess mammalian processing enzymes secrete active factor serum-free medium supplemented vWF stabilizing. Stable clones expressing B-domain-deleted FVIII CMV promoter purified monoclonal antibody immunoaffinity anion exchange preserving specific activity around 5000 IU per mg therapeutic use.

Ref: Blood Factor VIII CHO Production Kaufman 1988; FDA Recombinant Factor VIII Manufacturing Guidelines; Alberts Protein Glycosylation Mammalian Cells Chap 15.

View Details