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Lernkarten
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 ...)
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
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
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)
Eukaryotic expression systems: Filamentous fungi Advantages and Disadvantages
Advantages: Powerful secretory system, industrial-scale
Disadvantages: Complex genetics, lack of knowledge (physiology, biochemistry ...)
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
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
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
Cell-free expression systems Advantages and Disadvantages
Advantages: Extremly fast, incorporation of modified amino acids (fluorescence, biotin, radioactivity, heavy atoms, ...), lower sensitivity to toxic proteins, easy manipulation of reaction conditions, higher purity
Disadvantages: Small amounts of protein, expensive (based on commercial kits), only small scale
