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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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Recombinant protein production in eukaryotic cells l.jpg

Recombinant protein production in Eukaryotic cells

Dr. W. McLaughlin

BC35C


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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


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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


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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


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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)


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


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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


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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.


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S. cerevisiae expression vectors

  • Episomal, or plasmid vectors

  • Integrating vectors

  • Yeast artificial chromosomes (YACs)


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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


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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)


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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


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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


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YAC cloning system


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Recombinant Proteins- S. cerevisiae


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Direct expression in yeast

  • Produces proteins that accumulate in the cytoplasm

  • Production of human enzyme superoxide dismutase


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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


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S. cerevisiae expression vector


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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


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Purification of proteins

  • Including an export signal in-frame with His-tag

  • Purified by Ni-NTA affinity chromatography


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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


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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


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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


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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


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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


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Baculovirus system

  • Heterologous gene-expression levels can vary approx 1000-fold

  • Depends on the intrinsic nature of the gene and encoded protein


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Recombinant protein produced by baculovirus


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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


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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


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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


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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


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Reference

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


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