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Recombinant protein production in Eukaryotic cells

Recombinant protein production in Eukaryotic cells. Dr. W. McLaughlin BC35C. Recombinant protein production in Eukaryotic cells. rHuman protein must be identical to the natural protein

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Recombinant protein production in Eukaryotic cells

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  1. Recombinant protein production in Eukaryotic cells Dr. W. McLaughlin BC35C

  2. Recombinant protein production in Eukaryotic cells • rHuman protein must be identical to the natural protein • Prokaryotes are generally unable to produce authentic eukaryotic proteins due to the absence of appropriate mechanisms for carrying out the necessary post-translational modification to the protein

  3. Post-translational Modification • Correct disulphide bond formation. Reaction mediated by the enzyme disulphide isomerase. An improperly folded protein is unstable and lacks activity  • Proteolytic cleavage of a precursor form. Selected segments of amino acid sequences are removed to yield a functional protein

  4. Post-translational Modification • Glycosylation. Gives a protein with stability and, in some instances, its distinctive properties. The most common protein glycosylations occur by the addition of specific sugar residues to serine or threonine (O-linked) or to asparagine (N-linked)  • Addition of amino acids within proteins. Modification of this type includes phosphorylation, acetylation, sulfation

  5. Eukaryotic expression vectors • A selectable eukaryotic marker gene • A eukaryotic promoter sequence • The appropriate eukaryotic transcriptional and translational stop signal • A sequence that signals polyadenylation of the transcript messenger RNA (mRNA)

  6. p=promoter, t=termination & polyadenylation sequence, cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  7. Saccharomyces cerevisiae • A single cell • Well characterized genetically and physiologically • Can be readily grown in both small vessels and large scale bioreactors • Several strong promoters have been isolated and characterized

  8. Advantages of Yeast expression system • Carry out many post-translational modifications (phosphorylation, glycosylation and targeting) • Readily grown in small and large scale bioreactors • secretes few proteins, the product can easily be purified • generally recognized as safe (GRAS) • extensive screening of products is not required.

  9. S. cerevisiae expression vectors • Episomal, or plasmid vectors • Integrating vectors • Yeast artificial chromosomes (YACs)

  10. Yeast expression vector • Most widely used are the E. coli/yeast shuttle plasmids • Mitotically stabilized by autonomously replicating sequences – ARS/CEN region, 2 locus or by integration into the yeast genome

  11. Episomal vectors • Introduced by transformation into competent cells or electroporation • Used extensively for the production of heterologous proteins • Such plasmid-based expression systems are often unstable under conditions of large scale growth (>10 litres)

  12. YAC • The YAC is designed to clone large fragments of DNA (100 kb) • The YAC is maintained as a separate chromosome in the host yeast cell • The YAC is highly stable

  13. USES of YACs • the physical mapping of human genomic DNA • the analysis of large transcription units • the formation of genomic libraries containing DNA from individual human chromosomes

  14. YAC cloning system

  15. Recombinant Proteins- S. cerevisiae

  16. Direct expression in yeast • Produces proteins that accumulate in the cytoplasm • Production of human enzyme superoxide dismutase

  17. Heterologous gene expression in S. cerevisiae • Clone the human Cu/Zn-SOD cDNA into yeast episomal expression vector to obtain this authentic enzyme. • The cDNA is used as yeast cells do not remove introns. • If this was done in an E. coli vector there would be a problem with post transcription modification as the E. coli host cell only removes the initiator N-terminal methionine f-met from the Cu/Zn-SOD protein and the next amino acid alanine is not actylated

  18. S. cerevisiae expression vector

  19. Secretion of heterologous proteins • In yeast only secreted proteins are glycosylated • Facilitated by pre pro--factor or leader peptide • Active proteins are released to the extracellular environment • Leader peptide is removed by a yeast endoprotease

  20. Purification of proteins • Including an export signal in-frame with His-tag • Purified by Ni-NTA affinity chromatography

  21. Limitations of Yeast expression systems • Loss of plasmid even when inducible promoters are used • The heterologous protein is often hyperglycosylated > 100 mannose residues • Protein retained within the periplasmic space, this makes it difficult to purify the protein

  22. Cultured Insect Cell Expression system • Baculovirus vectors used to heterologously express proteins in insect cells • Based on the ability of the virus to infect and multiply in cultured insect cells

  23. Baculovirus vectors • Most widely used virus Autographa californica nuclear polyhedrosis virus (AcNPV) • A lytic virus that infects lepidopterans • E.g. the fall armyworm, Spodoptera frugiperda cell lines Sf9 and Sf21

  24. Advantages of insect cells • Recognizes most vertebrate protein-targeting sequences and thus express a wide variety proteins • Many post-translational modification (phoshorylation, glycosylation, precursor processing, and targeting) • Recombinant protein can either be produced within the cell or secreted into the culture medium

  25. Baculovirus Expression Vector System • Foreign gene cloned into a transfer vector based on E. coli plasmid that carries a segment of the DNA from AcNPV • Co-transfected along with ds-baculovirus DNA into insect cells • Homologous recombination of the transfer vector with insert DNA with viral genome leads to the cloned gene being transferred into the AcNPV DNA. •  Heterologous proteins after 4-5 days

  26. Baculovirus system • Heterologous gene-expression levels can vary approx 1000-fold • Depends on the intrinsic nature of the gene and encoded protein

  27. Recombinant protein produced by baculovirus

  28. Mammalian cells • Mammalian cells the best host for the expression or recombinant vertebrate proteins • Produce the same post-translational modifications and recognize the same signals • Expression levels are usually low

  29. Mammalian cell expression Vectors • Contain an efficient promoter elements for high level transcriptional initiation • Contain mRNA processing signals • Contain selectable markers • Plasmid sequences for propagation in bacterial hosts

  30. Mammalian cell expression Vectors • contains a eukaryotic origin of replication from an animal virus, e.g. Simian virus 40 (SV40) • Origin of replication from E. coli • promoter sequences that drive both the cloned gene(s) and the selectable marker gene(s) • transcription termination sequences - adenylation signals from animal virus e.g. SV40

  31. Mammalian expression vectors • Versatile and effective • Used for the production of authentic recombinant proteins for research and clinical trials • Industrial production using engineered mammalian cells is costly

  32. Reference • Molecular Biotechnology: Principles and applications of recombinant DNA. Glick and Pasternak 2nd edition. Chapter 7

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