Difference between revisions of "Team:Freiburg/Project/Cellfree Expression"
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<h3 class="sectionedit2">Why Do We Use Cell-free Expression?</h3> | <h3 class="sectionedit2">Why Do We Use Cell-free Expression?</h3> | ||
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<h3 id="diamix" class="sectionedit3">Basics of Cell-free Expression</h3> | <h3 id="diamix" class="sectionedit3">Basics of Cell-free Expression</h3> | ||
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Two basic components are needed to conduct in vitro protein expression: | Two basic components are needed to conduct in vitro protein expression: | ||
<ul> | <ul> | ||
− | <li | + | <li >the genetic template (mRNA or DNA) encoding the target protein</div> |
</li> | </li> | ||
− | <li | + | <li>a reaction solution containing the necessary transcriptional and translational molecular machinery </div> |
</li> | </li> | ||
</ul> | </ul> | ||
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+ | Cell extracts supply all or most of the molecules of the reaction solution, including: | ||
+ | <ul> | ||
+ | <li >RNA polymerases for mRNA transcription</div> | ||
+ | </li> | ||
+ | <li>ribosomes for polypeptide translation </div> | ||
+ | </li> | ||
+ | <li>tRNA and amino acids</div> | ||
+ | </li> | ||
+ | <li> enzymatic cofactors and an energy source</div> | ||
+ | </li> | ||
+ | <li>cellular components essential for proper protein folding </div> | ||
+ | </li> | ||
+ | </ul> | ||
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+ | Normally, Cell lysate already contain most of the components needed for cell free expression.The machinery that normally conducts Translation and Transcription of various proteins in the organism can be "reprogrammed" to produce only the protein of our choice. Our building blocks, the aminoacids and NTPs are already present but are also added to increase the efficiency of our system and to compensate for the missinf energy regeneration system. | ||
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Cell lysates provide the correct composition and proportion of enzymes and building blocks required for translation. (Usually, an energy source and amino acids must also be added to sustain synthesis.) Cell membranes are removed to leave only the cytosolic and organelle components of the cell (hence the term, “cell-free extracts”). The first types of lysates developed for cell-free protein expression were derived from prokaryotic organisms. More recently, systems based on extracts from insect cells, mammalian cells and human cells have been developed and made commercially available. | Cell lysates provide the correct composition and proportion of enzymes and building blocks required for translation. (Usually, an energy source and amino acids must also be added to sustain synthesis.) Cell membranes are removed to leave only the cytosolic and organelle components of the cell (hence the term, “cell-free extracts”). The first types of lysates developed for cell-free protein expression were derived from prokaryotic organisms. More recently, systems based on extracts from insect cells, mammalian cells and human cells have been developed and made commercially available. |
Revision as of 23:45, 16 September 2015
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Methodology Cell-free Expression
Why Do We Use Cell-free Expression?
Cell-based protein expression is a well established method to obtain large amounts of a target protein. It enables accumulation and purification of the protein in quantities sufficient for various in vitro applications. Nonetheless, it is a tedious task to generate all the genetically modified organisms if a variety of proteins needs to be expressed. Additionally, purification of a distinct protein can not be performed according to a generalized protocol, but has to be optimized for every protein separately. Cell-free expression represents a possibility to overcome many challenges of conventional protein expression and offers many advantages concerning our project in particular.1) Generally speaking, it saves a lot of time and money to avoid the generation of genetically modified organisms for every protein. Suitable DNA sequences are constructed once, for example by having it synthesized by a company, and can be stored until needed. In terms of purification, cell-free expression avoids the need for cell lysis and therefore circumvents this harsh procedure, thus preserving the protein. In cell-based expression, too strong induction often results in aggregated and therefore non-functional protein. This risk is minimized by using cell-free expression, since the expressed protein is dispersed in a far larger volume than the intracellular space.2) For translating DNA templates into protein microarrays in a microfluidic set-up, cell-free expression is the method of choice. This system is capable of expressing many different sequences at once. Additionally, the microfluidic setup provides the opportunity to constantly supplement the expression. Replacing depleted components, like dNTPs, amino acids or energy sources like creatine phosphate, enables higher yields of protein.
Basics of Cell-free Expression
Two basic components are needed to conduct in vitro protein expression:
- the genetic template (mRNA or DNA) encoding the target protein
Cell extracts supply all or most of the molecules of the reaction solution, including:
- RNA polymerases for mRNA transcription