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Sprache English
Stufe Universität
Erstellt / Aktualisiert 07.06.2021 / 08.06.2021
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Factors to consider while choosing the appropriate expression system

-What type of protein do I want to express? (Origin, subcellular localization, ...)

-For what purpose? (Activity assay, "in vivo" functional studies, medical ...)

-How much protein I need= (industrial vs lab scale)

-Does my protein need post-translational modifications/ proper folding for strcture/activity? (Glycosylation, phosphorylation...)

-Do I have the appropriate facilities/equipment? (Safety hazard, culture requirements ...)

-Other factors (cost, speed, scalability, quality ...)

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Prokaryotic expression systems Advantages and Disadvantages

Advantages: Very high expression levels, low cost, simple culture conditions, fast cell multiplication, relative scalability

Disadvantages: Minimal post-translational modifications, inefficent disulfide bond formation, poor protein folding, 

codon usage different to eukaryotes, inclusion body formation, potential endotoxin contamination

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Eukaryotic expression systems Advantages and Disadvantages

Advantages: Possible post-transcriptional modifications, correct protein folding, avoid co-production of toxic compounds

Disadvantages: In general more expensive than prokaryotic systems, complex growth requirements, limited scalabilty

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Eukaryotic expression systems: Yeast. Advantages and Disadvantages

Combines advantages from prokaryotes: high expression, fast growth, easy maintenance, easy scale-up, cheap

with advantages from eukaryotes: Able to perform most eukaryotic poast-translational modifications, protein folding, absence of endotoxins

Disadvantages: Glycosylation still different to mammals and plants, protein hyperglycosylation (N-glycan strutctures allergenic)

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Eukaryotic expression systems: Filamentous fungi Advantages and Disadvantages

Advantages: Powerful secretory system, industrial-scale

Disadvantages: Complex genetics, lack of knowledge (physiology, biochemistry ...)

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Eukaryotic expression systems: Mammalian cells Advantages and Disadvantages

Advantages: Proper protein folding, proper post-translational modifications, several cell lines available

 

Disadvantages: Expresive culture media, slow growth, complicated technology, potential contamination animal viruses

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Eukaryotic expression systems: Insect cells Advantages and Disadvantages

Advantages: Extensive post-translational modifications, liquid culture compatible (several stablished lines available), baculoviruses have very restricted host range (non infectious for vertebrates), no need of helper viruses, thousands of high-value proteins industrially produced, e.g. vaccines, gene therapy, MultiBac- advanced baculovirus system for multiprotein complexes

Disadvantages: Expensive culture media, large volumes of virus needed, infected cells eventually die, no continous production, still different glycosylation pattern - "Humanized" insect cell lines expressing glycosylation machinery

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Eukaryotic expression systems: Plants Advantages and Disadvantages

Advantages: simple and inexpensive growth requirements, high biomass production (scalability), post-translational modification/processing, multimeric protein complexes, organ and development-specific promoters available

Disadvantages: variable yield, contamination with pesticides/fertilizers, impact of pests/diseases