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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