Recombinant protein production in eukaryotic cells
Download
1 / 34

Recombinant protein production in Eukaryotic cells - PowerPoint PPT Presentation


  • 1192 Views
  • Updated On :

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Recombinant protein production in Eukaryotic cells' - cole


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

Recombinant protein production in eukaryotic cells2 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg

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


Saccharomyces cerevisiae l.jpg
Saccharomyces cerevisiae cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Advantages of Yeast expression system cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
S. cerevisiae cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication expression vectors

  • Episomal, or plasmid vectors

  • Integrating vectors

  • Yeast artificial chromosomes (YACs)


Yeast expression vector l.jpg
Yeast expression vector cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Episomal vectors cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
YAC cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
USES of YACs cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • the physical mapping of human genomic DNA

  • the analysis of large transcription units

  • the formation of genomic libraries containing DNA from individual human chromosomes


Slide14 l.jpg

YAC cloning system cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication


Recombinant proteins s cerevisiae l.jpg
Recombinant Proteins- cs=cloning site, EMS=eukaryotic marker system, ori=origin of replicationS. cerevisiae


Direct expression in yeast l.jpg
Direct expression in yeast cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • Produces proteins that accumulate in the cytoplasm

  • Production of human enzyme superoxide dismutase


Heterologous gene expression in s cerevisiae l.jpg
Heterologous gene expression in S. cerevisiae cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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


S cerevisiae expression vector l.jpg
S. cerevisiae expression vector cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication


Secretion of heterologous proteins l.jpg
Secretion of heterologous proteins cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Purification of proteins cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

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

  • Purified by Ni-NTA affinity chromatography


Limitations of yeast expression systems l.jpg
Limitations of Yeast expression systems cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Cultured Insect Cell Expression system cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Baculovirus vectors cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Advantages of insect cells cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Baculovirus Expression Vector System cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Baculovirus system cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

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

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


Recombinant protein produced by baculovirus l.jpg
Recombinant protein produced by baculovirus cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication


Mammalian cells l.jpg
Mammalian cells cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Mammalian cell expression Vectors cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Mammalian cell expression Vectors cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Mammalian expression vectors cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

  • 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 l.jpg
Reference cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

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


ad