Simplified method makes cell-free protein synthesis more flexible and more accessible



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PICTURE

PICTURE: A cartoon scheme of cell-free protein synthesis, a biotechnology that exploits the genetic code of the cell in a test tube. The diagram represents a simplified approach that allows scientists to perform …
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Credit: Nicole E. Gregorio / Cal Poly San Luis Obispo

Researchers radically simplified the cell-free protein synthesis method (CFPS), a technique that could become fundamental to medical research.

Protein synthesis is essential for many types of pharmaceutical and genetic research. For years, proteins could only be synthesized in living cells. CFPS offers the new biosynthetic capacity of proteins in a test tube in a few hours without resorting to living cells. This process offers a new level of control over protein production, an incredible benefit for researchers pursuing high throughput testing, biosensor construction, metabolic engineering, and so on.

"This biotechnology exploits the genetic code in a test tube, providing direct access to the biological machinery traditionally locked in the cell," said Javin Oza, professor of biochemistry at Cal Poly, San Luis Obispo. "This allows scientists and engineers to manufacture vaccines and therapeutic proteins, as well as perform diagnostic tests on demand, in the laboratory or in the field.In class, the CFPS allows students to become familiar with with the genetic code in a way based on surveys. "

Although the acceptance of the CFPS as a promising technology has increased significantly over the past two decades, it still suffers from some limitations due to the difficulty and costs involved in implementing the technique.

As reported in Journal of Visualized Experimentation, a group of Cal Poly researchers led by Oza and his collaborator Katharine Watts have developed a method to make the CFPS widely accessible. The main advantages of the new technique are speed, cost-effectiveness and a much less complex reaction configuration compared to other CFPS systems. In order to further reduce the obstacles preventing scientists from adopting this method, the publication includes a video guide for the implementation of the new procedure.

"This protocol simplifies and clarifies methods for implementing cell-free protein synthesis by non-experts," Oza said. "Improving access to these methods will help democratize the platform and its wide range of applications."

The new technique requires only basic laboratory training so that new users can implement CFPS in their laboratories, from preparation of growth and cell extract to in vitro protein synthesis reactions. Researchers have developed reagent premixes that significantly reduce the risk of error during setup and increase the likelihood of a successful reaction. These chemical and biological reagents are stable and can withstand several freeze / thaw cycles. In the paper, researchers also identify and report variables that have a significant impact on the success of CFPS implementation and the variables that new users need to optimize for successful responses.

The simplified procedure is functionally comparable to the more technical methods previously used and can be used to screen protein products more rapidly, which greatly increases the number of tests that can be performed over a period of time. This capability supports sophisticated research efforts, such as functional genomics and metabolic engineering.

"In our collaboration, we use the simplified CFPS method to synthesize and design complex mega-enzymes, which are traditionally difficult to express," said Watts, also professor of biochemistry at Cal Poly. "The use of CFPS allows much faster design-build-test cycles for the engineering of these mega-enzymes."

In addition to primary research, the new CFPS method can be used as a teaching tool in high school and undergraduate classes. Once packaged for educational use, it offers students the opportunity to explore genetics in a hands-on learning environment.

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This research was funded by the National Science Foundation.

Reference: Levine, M. Z., Gregorio, N.E., Jewett, M.C., Watts, K.R., Oza, J.P. Synthesis of Escherichia Coli-based Cell-free Proteins: Protocols for Robust, Flexible and Accessible Platform Technology. J. Vis. Exp. e58882, doi: 10,3791 / 58882 (2019).

Cal Poly College of Science and Mathematics offers intensive laboratory training in a favorable atmosphere. Undergraduate research is at the heart of the Learning Through College approach, as it allows students to work closely with faculty mentors on concrete research projects. The college houses undergraduate and postgraduate teacher education programs, providing prospective state teachers with a solid background in science, engineering, technology and mathematics (STEM). It is one of the six poly-technical colleges of Cal Poly, a four-year-old comprehensive polytechnic university located in San Luis Obispo, California.

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