Pyruvylated Lactose-based Cell-free System Construction Service
The Cost-effective Solution for Pyruvylated Lactose-based Cell-free System Construction
CD BioGlyco provides the pyruvylated lactose-based cell-free system construction service, providing a cost-effective solution for protein synthesis. Using pyruvylated lactose as an energy source, this system is versatile for various applications. The system facilitates protein production quantification and the regeneration of nucleotides and pyruvate during reactions. With our service, researchers harness this innovative system for efficient and economical protein synthesis in cell-free expression systems.
Fig.1 The process of pyruvylated lactose-based cell-free system construction. (CD BioGlyco)
- Begin by preparing a cell-free extract from a suitable host organism, such as Escherichia coli. This involves lysing the cells and removing cellular debris to obtain the extract containing the necessary components for protein synthesis.
- Modify the cell-free extract to enable the synthesis of pyruvylated lactose by adding or overexpressing specific enzymes or pathways involved in its biosynthesis.
- Ensure the presence of necessary substrates for pyruvylated lactose synthesis in the cell-free system, including lactose, pyruvate, and other cofactors or precursors required for enzymatic reactions.
- Determine optimal conditions for the cell-free synthesis of pyruvylated lactose by optimizing factors like temperature, pH, and reaction time to maximize enzymatic reaction efficiency.
- Track the formation of pyruvylated lactose during the synthesis process using methods like high-performance liquid chromatography (HPLC) or mass spectrometry (MS) to measure the yield of the product accurately.
- Scale up the reaction to produce larger quantities of pyruvylated lactose once the cell-free system is established successfully. Purify the synthesized pyruvylated lactose using chromatography or filtration techniques to obtain a pure product.
Publication Data
DOI: https://doi.org/10.1021/acssynbio.1c00342
Journal: ACS Synthetic Biology
IF: 4.7
Published: 2022
Technology: Drying systems, Cell-free protein synthesis
Results: The research in this article focuses on reducing the cost of constructing cell-free expression systems. The authors developed three novel approaches to reduce costs. Initially, the researchers assessed the effectiveness of silica beads in combination with a slight vacuum to desiccate cell-free constituents, employing sugars as lyoprotectants. This method sustained protein synthesis capacity at 45% and 75% after a two-week storage period at room temperature, utilizing various energy sources. Subsequently, they observed that the inclusion of lactose improved cell-free reactions, resulting in a 188% rise in protein yield when maltodextrin served as the energy source. This strategy yielded comparable outcomes to conventional energy sources. Lastly, they noted the continued endogenous biosynthesis of nucleoside mono- and triphosphates in cell-free reactions reliant on phosphoenolpyruvate (PEP). This means that reactions could be composed without the addition of nucleotides or nucleoside monophosphates, further reducing costs.
Fig.2 Constructing cell-free expression systems for low-cost access. (Guzman-Chavez, et al., 2022)
Applications of Pyruvylated Lactose-based Cell-free System
- The pyruvylated lactose-based cell-free system offers a cost-effective approach to protein synthesis. By using pyruvylated lactose as an energy source, the system eliminates the need for expensive substrates like PEP or adenosine triphosphate (ATP). This makes it an attractive option for low-budget protein production.
- The cell-free system can be used to produce high-value protein reagents, such as enzymes or diagnostic proteins, at a lower cost compared to traditional methods. For example, the system has been used to produce Bst DNA polymerase, which is commonly used in loop-mediated isothermal amplification (LAMP) assays. The protein produced using the pyruvylated lactose-based system showed equal effectiveness to the commercially available version but at a significantly reduced cost.
- The system can be utilized for the production of toxic proteins, such as type IIS restriction enzymes like BsaI, which are commonly used in Golden Gate cloning. These enzymes are difficult to obtain in large quantities using traditional methods due to their toxicity. However, the pyruvylated lactose-based system allows for the production of these enzymes using chemically synthesized linear DNA templates, eliminating the need for specialized E. coli strains or plasmid DNA templates.
- The addition of lactose to the cell-free system can improve protein yields when using linear DNA templates. Linear DNA templates are a popular alternative to plasmids due to their ease of preparation. However, they are prone to degradation by endogenous exonuclease activity. The pyruvylated lactose-based system, supplemented with lactose, helps stabilize linear DNA templates, leading to improved protein synthesis yields.
Advantages
- CD BioGlyco understands the importance of cost-effectiveness in research and development processes. We strive to offer efficient solutions that optimize resources without compromising on quality or performance.
- CD BioGlyco maintains stringent quality control measures throughout the construction process of pyruvylated lactose-based cell-free systems. This ensures the reliability and reproducibility of the systems delivered to clients.
- CD BioGlyco is committed to delivering projects within stipulated timelines. Our efficient project management ensures that clients receive their pyruvylated lactose-based cell-free systems promptly, facilitating the smooth progression of research activities.
Similar Services at CD BioGlyco
CD BioGlyco adopts innovative approaches and stays abreast of the latest advancements in synthetic biology. Based on our powerful GlycoChas™ Cells services, we provide the custom Cell-free System Construction Service for our clients according to their needs and we also would like to work with clients on Chassis Cell Development to produce more glycan-related products in a highly effective way. Please do not hesitate to contact us if you are interested in our services.
Reference
- Guzman-Chavez, F.; et al. Constructing cell-free expression systems for low-cost access. ACS Synthetic Biology. 2022, 11(3): 1114-1128.
For research use only. Not intended for any clinical use.
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