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#recombinant protein

3 public questions tagged with this topic.

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.

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Gene Cloning Process

What change improves protein solubility in E. coli expression systems?

Lowering post-induction temperature to 16-25°C markedly improves solubility of recombinant proteins expressed in Escherichia coli. Reduced temperature slows transcription and translation rates, decreasing nascent chain concentration and allowing more time for co-translational folding assisted by chaperones GroEL and DnaK. Hydrophobic interactions driving aggregation are temperature dependent and weakened at lower temperatures, thus inclusion body formation is minimized. High IPTG or high temperature exacerbates aggregation, while glucose suppresses induction via catabolite repression but does not enhance folding quality of already induced protein.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Cloning Process

Insoluble expression of protein X at 37°C in E. coli is due to:

High-level expression of heterologous proteins at 37°C in Escherichia coli under strong promoters like T7 drives rapid polypeptide synthesis exceeding the capacity of endogenous chaperone systems such as GroEL-GroES and DnaK. Hydrophobic stretches improperly exposed interact intermolecularly, forming insoluble aggregates termed inclusion bodies sequestered in cytoplasm. Although protein is abundant, it is misfolded and functionally inactive. This aggregation is temperature and induction rate dependent rather than due to promoter absence, inducer failure, or cytotoxicity, and can be mitigated by lowering culture temperature or using solubility tags.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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