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Protein Expression

Protein expression systems

Protein expression is the biotechnological process of producing a target protein. It is typically achieved by the manipulation of gene expression in an organism, which expresses a large amount of a recombinant gene. This includes the transcription of the recombinant DNA to mRNA, the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

Commonly used protein production systems include in vitro (Cell-free) and in vitro (Cell-based) Protein Expression systems:

Cell-free systems

Cell-free production of proteins is performed in vitro using purified RNA polymerase, ribosomes, tRNA and ribonucleotides. These reagents may be produced by extraction from cells or from a cell-based expression system. Due to the low expression levels and high cost of cell-free systems, cell-based systems are more widely used.

Cell-based systems

The oldest and most widely used expression systems are cell-based and may be defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level".

There are many different host cells may be used for expression. For example, common hosts are bacteria (such as E.coli, B. subtilis), yeast (such as S.cerevisiae) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are viruses (such as baculovirus, retrovirus, adenovirus), plasmids, artificial chromosomes and bacteriophage (such as lambda). The best expression system depends on the gene involved. Insect or mammal cell lines are used when human-like splicing of mRNA is required. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography, Electron Microscope or Nuclear Magnetic Resonance experiments for structure determination.

Expression host comparison

Recombinant protein expression technology enables various downstream applications, including analysis of gene regulation, protein structure and function, protein-protein interactions and drug discovery and development. Using the right protein expression system for your specific application can be critical for success.

Host system Advantages Challenges
In vitro (Cell-Free)
  • Scalable
  • Simple format
  • Rapid expression directly from plasmid
  • Open system—easily add components to enhance solubility or functionality
  • Large-scale expression > 3 mg
Bacterial
  • Scalable
  • Low cost
  • Simple culture conditions
  • Protein solubility
  • Minimal posttranslational modifications
  • May be difficult to express functional mammalian proteins
Yeast
  • Eukaryotic protein processing
  • Scalable up to fermentation (grams per liter)
  • Simple media requirements
  • Fermentation required for very high yields
  • Growth conditions may require optimization
Algae
  • Genetic modification and expression systems for photosynthetic microalgae
  • Superb experimental control for biofuel, nutraceuticals and specialty chemical production
  • Optimized system for robust selection and expression
Insect
  • Postranslational modifications similar to mammalian systems
  • Greater yield than mammalian systems
  • More demanding culture conditions
Mammalian
  • Highest level of correct post-translational modifications
  • Highest probability of obtaining fully functional human proteins
  • Multimilligram per liter yields only possible in suspension cultures
  • More demanding culture conditions

Reference:

  1. Gräslund, Susanne, et al. Protein production and purification. Nature Methods. Feb 2008, 5 (2): 135–146. doi:10.1038/nmeth.f.202
  2. Rosenblum, G; Cooperman, BS. Engine out of the chassis: cell-free protein synthesis and its uses. FEBS Lett. Jan 2014. 588 (2): 261-8. doi:10.1016/j.febslet.2013.10.016. PMID 24161673
  3. Definition: expression system. Online Medical Dictionary. Centre for Cancer Education, University of Newcastle upon Tyne: Cancerweb. 1997-11-13. Retrieved 2008-06-10
  4. Expression system - definition. Biology Online. Biology-Online.org. 2005-10-03. Retrieved 2008-06-10.