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Kartei Details

Zusammenfassung	This flashcard set delves into the intricacies of protein expression systems, comparing various platforms like cell-free, eukaryotic, and prokaryotic systems. It highlights the advantages and disadvantages of each, focusing on factors like glycosylation, scalability, and post-translational modifications. Ideal for university students and researchers, this set aids in understanding the complexities of protein production, helping them choose the right system based on specific needs like protein type, required modifications, and production scale.
Karten	9
Lernende	1
Sprache	English
Kategorie	Biologie
Stufe	Universität
Erstellt / Aktualisiert	07.06.2021 / 08.06.2021
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Factors to consider while choosing the appropriate expression system

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-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

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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

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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

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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

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Advantages: Powerful secretory system, industrial-scale

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

Eukaryotic expression systems: Mammalian cells Advantages and Disadvantages

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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

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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

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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

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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

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