recombinant protein production in eukaryotic cells
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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 cells2
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
post translational modification
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
post translational modification4
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
eukaryotic expression vectors
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)
slide6

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

saccharomyces cerevisiae
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
advantages of yeast expression system
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.
s cerevisiae expression vectors
S. cerevisiae expression vectors
  • Episomal, or plasmid vectors
  • Integrating vectors
  • Yeast artificial chromosomes (YACs)
yeast expression vector
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
episomal vectors
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)
slide12
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
uses of yacs
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
direct expression in yeast
Direct expression in yeast
  • Produces proteins that accumulate in the cytoplasm
  • Production of human enzyme superoxide dismutase
heterologous gene expression in s cerevisiae
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
secretion of heterologous proteins
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
purification of proteins
Purification of proteins
  • Including an export signal in-frame with His-tag
  • Purified by Ni-NTA affinity chromatography
limitations of yeast expression systems
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
cultured insect cell expression system
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
baculovirus vectors
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
advantages of insect cells
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
baculovirus expression vector system
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
baculovirus system
Baculovirus system
  • Heterologous gene-expression levels can vary approx 1000-fold
  • Depends on the intrinsic nature of the gene and encoded protein
mammalian cells
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
mammalian cell expression vectors
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
mammalian cell expression vectors31
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
mammalian expression vectors
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
reference
Reference
  • Molecular Biotechnology: Principles and applications of recombinant DNA. Glick and Pasternak 2nd edition. Chapter 7
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