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Target gene

Expression plasmids can be unstable in the bacterial host strain. Stability can be increased by integrating the construct into the chromosome. Target gene. Promoter. Integration sequence. Antibiotic resistance gene. Chromosome.

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Target gene

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  1. Expression plasmids can be unstable in the bacterial host strain. Stability can be increased by integrating the construct into the chromosome. Target gene Promoter Integration sequence Antibiotic resistance gene Chromosome

  2. The plasmid does not replicate in the host cell, but integrates into the chromosome by homologous recombination. Cells with an integrated plasmid are antibiotic resistant. The integration sequence can be any non-essential region of the chromosome. The integration event duplicates the insertion site in the chromosome.

  3. DNA slippage during replication DNA sequences between direct repeats can become duplicated. 3’

  4. If antibiotic selection is maintained, several duplications can occur.

  5. In the absence of antibiotic selection, recombination between direct repeats can excise the plasmid (reverse the integration event).

  6. Overproduction of eukaryotic proteins Many eukaryotic proteins are modified after translation. Post-translational modifications: 1. Disulphide bond formation. 2. Proteolytic cleavage of a precursor. 3. Glycosylation. 4. Additions to amino acids within proteins. Bacterial host strains cannot carry out these modifications (especially 3 and 4).

  7. Protein glycosylation In eukaryotic cells, membrane or secreted proteins may be glycosylated. O-linked oligosaccharides are linked to SER or THR and are very heterogeneous. N-linked oligosaccharides are linked to ASN residues within the sequence ASN-X-SER (X  PRO).

  8. MAN MAN 1,3 1,6 MAN b1,4 GLC NAc b1,4 GLC NAc ASN N-linked oligosaccharides contain a common core consisting of 2 N-acetyl glucosamine and 3 mannose residues.

  9. Additional sugars are added to the core to form a huge variety of oligosaccharide structures. Several mannose residues are added to form the high mannosetype. In the complex type, different combinations of N-acetyl glucosamine, galactose, sialic and fucose residues are added. The oligosaccharides are heterogeneous in vivo.

  10. Glycosylation affects folding, stability, solubility, immunogenicity, biological activity, and in vivo clearance rate. Some non-glycosylated recombinant proteins retain activity. Some human proteins must be glycosylated to be effective e. g. erythropoietin and clotting factor IX. These proteins have to be overproduced in eukaryotic cells.

  11. Eukaryotic expression vectors have been developed for yeast, insect and mammalian cells. Most are bifunctional and have origins of replication and selectable markers for E. coli and the eukaryotic cell.

  12. Maintained by LEU-requiring host cells S. cerevisiae expression vectors have been based on the 2 micron circle(a naturally occurring yeast plasmid). AmpR ori E Leu 2 gene Polyadenylation signal Strong yeast promoter Multiple cloning site

  13. Only secreted proteins are glycosylated. Yeast signal peptide is genetically fused to foreign gene via codons for LYS-ARG. Yeast signal peptidase recognises the LYS-ARG and releases the mature protein after export.

  14. Baculoviruses - DNA viruses that infect insects. AcMNPV virus has been used to develop an expression vector. This is grown on insect cell lines. Infected cells give single virions and polyhedrons, several virions trapped in a matrix of polyhedrin protein. Polyhedrin is synthesised in massive quantities over 4 to 5 days. Recombinant baculoviruses overexpress foreign genes from the polyhedrin promoter.

  15. Partially dissolved polyhedron containing bundles of virus particles. Polyhedra under light microscope

  16. Bombyx mori nuclear polyhedrosis virus. Individual baculovirus particles

  17. The target gene is introduced into the baculovirus genome by homologous recombination. Transfer vector Foreign gene Polyhedrin gene Baculovirus genome

  18. Recombinant baculoviruses can be identified by PCR. These give protein overproduction in cultured insect cells or whole insect larvae. Insect cells have the glycosyltransferases for correct glycosylation of human proteins.

  19. Caterpillars infected with recombinant baculovirus. During infection, the interior of each larva liquefies. High levels of recombinant protein accumulate.

  20. Mammalian expression systems Mammalian cells do not contain plasmids Some animal virus genomes can be modified to exist as extrachromosomal elements in cultured cells e. g. the human papova BK virus.

  21. Neomycin G418 increases copy number to 8000 per cell BKV replication genes neoR ori BKV oriE DRE MMTP AmpR Dioxin-responsive enhancer Mouse mammary tumour promoter

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