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.