Empower Cell-free Systems with Mono- or Di-Fucosylated LacNAc at CD BioGlyco

Glycosylation plays an important role in cells and confers beneficial properties to protein therapeutics. Glycoproteins can be efficiently produced and engineered by constructing to identify viable glycosylation pathways. As a specialized biology research company, CD BioGlyco constructs a versatile Cell-free System based on in-depth biological knowledge and best-in-class biological technologies and provides clients with a trusted mono- or di-fucosylated LacNAc-based cell-free system construction service.

Establishment of a cell-free platform

We carry out the assembly of the cell-free platform by rapid in vitro mixing and expression. In this process, we assemble the glycosylation pathway by mixing and matching extracellularly synthesized glycosyltransferases and expanding glucose primers installed by N-glycosyltransferases on protein targets.

We mount a glucose primer onto the protein by N-glycosyltransferase, which can then be sequentially elaborated by co-expressed glycosyltransferases. To enhance glycosyltransferase expression, we add the glycosyltransferase cleavage required for complete glycoprotein conversion to each glycosylation reaction.

We use glycosylated sequences with individual optimizations as model target proteins and test their full-length expression using sodium dodecyl sulfate-polyacrylamide gel electrophoresis radioautography. We purify the substrates using functionalized magnetic beads and characterize the resulting glycoproteins by liquid chromatography-mass spectrometry.

Mono- or di-fucosylated LacNAc modular construction

We screen different types of fucosyltransferases from Helicobacter pylori, which in turn construct fucosylated LacNAc. Next, we explore the biosynthetic pathway of N-linked single or double fucosylated LacNAc. We use bacterial fucosyltransferases as a mediator to install fucosylation sequences to LacNAc.

Fig.1 Construct a cell-free system using mono- or di-fucosylated LacNAc. (CD BioGlyco)

Frequently Asked Questions

• What is a cell-free system?
  A cell-free system utilizes cellular extracts (rather than the cells themselves) for gene transcription and translation. It consists of three main components:
  
  • Cell extract: This is a mixture of extracts from cells that contain biomolecules within the cell, such as nucleic acids, proteins, enzymes, etc.
  • Reaction mixture: A mixture used to drive gene expression in vitro, usually including the desired cellular components and other necessary reagents.
  • Nucleic acid template: Acts as a starting point for gene expression and can be either DNA or RNA.
• What are the advantages of cell-free systems?
  • Cell-free systems are not dependent on living cells and are therefore suitable for specific research needs such as the expression of toxic target proteins.
  • Cell-free systems can also overcome some of the disadvantages of in vivo expression systems, such as the toxicity of overexpression products to host cells, insolubility of the products, and rapid degradation of the proteins.

Advantages

• Glycosylation technology: We are proficient in glycosylation reactions and have a deep knowledge of glycochemistry to Produce or handle complex glycan molecules.
• Glycomics analysis: We have a state-of-the-art glycosyl-based Chassis Cell Platform that allows for the Development of Glycosyl-based Chassis Cells and provides complex glycomics analysis.
• Technical consulting: We provide professional consulting on glycochemistry, glycobiology, and glycopharmaceuticals to help clients solve technical problems.

CD BioGlyco is a company with extensive experience in the field of synthetic biology. We have expertise in the construction of cell-free systems and provide our clients with dedicated cell-free system construction services. Please feel free to contact us and we will be happy to answer them if you have any questions.

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