Gene to Protein Synthesis Custom Order

Performing gene to protein synthesis in a cell-free system offers a unique approach compared to relying on living cells. Here's a breakdown of the key steps involved:

• 1. Preparation of Cell-Free Extract:
  • The first step involves creating a cell-free extract. This is essentially a cellular lysate, a suspension made by breaking open cells and removing membranes and cellular debris.
  • The extract retains the essential machinery necessary for protein synthesis, including ribosomes, transfer RNAs (tRNAs), amino acids, and enzymes required for translation initiation, elongation, and termination.
  • Common sources for cell-free extracts include E. coli, wheat germ, and rabbit reticulocyte lysates, each with advantages and limitations depending on the desired protein.
• 2. DNA Template Acquisition:
  • You'll need a DNA template encoding the protein of interest. Here are two main approaches:
  • Plasmids: These circular DNA molecules can be engineered to contain the gene sequence along with regulatory elements for efficient transcription in the cell-free system. Plasmids are often used when large quantities of protein are needed.
  • PCR Products: PCR can be used to amplify a specific DNA fragment containing the desired gene sequence. This approach is faster than plasmid construction but might yield lower protein amounts.
• 3. In Vitro Transcription (IVT):
  • This step involves synthesizing mRNA from the DNA template. Purified RNA polymerase enzymes are introduced into the cell-free extract along with the DNA template and nucleoside triphosphates (the building blocks of RNA).
  • The RNA polymerase recognizes the promoter sequence on the DNA and uses it to transcribe the gene into a complementary mRNA molecule. This mRNA molecule carries the genetic information for protein synthesis.
• 4. Cell-Free Translation:
  • The synthesized mRNA is added to the cell-free extract containing all the necessary components for translation.
  • Ribosomes in the extract recognize the start codon (AUG) on the mRNA and initiate protein synthesis. tRNAs carrying specific amino acids interact with the mRNA codons, and the ribosome links the amino acids together according to the mRNA sequence.
  • This process continues until a stop codon is reached on the mRNA, signaling the release of the completed protein from the ribosome.

Advantages of Cell-Free Protein Synthesis:

• Speed and Simplicity: Cell-free systems offer a faster and more streamlined approach compared to traditional methods involving cell culture.
• Scalability: Protein production can be easily scaled up or down by adjusting the reaction volume and components.
• Purity: Proteins synthesized in cell-free systems are typically free of contaminants present in whole cells.
• Direct Manipulation: The system allows for easy manipulation of reaction conditions and incorporation of non-standard amino acids into the protein.

Applications of Cell-Free Protein Synthesis:

• Rapid Protein Production: This technique is valuable for research purposes, such as studying protein function or screening for protein activity.
• Membrane Protein Production: Cell-free systems can be advantageous for producing membrane proteins, which are often difficult to express in living cells.
• In Vitro Diagnostics: Cell-free protein synthesis can be used to develop diagnostic tools by expressing specific antigens for antibody detection.
• Biomanufacturing: This approach holds promise for the future production of complex proteins for various applications.

Bioneer's ExiProgen™ is a system specifically designed for automated protein synthesis and nucleic acid extraction. It simplifies and streamlines these processes within a research environment.

• ExiProgen utilizes cell-free protein synthesis. This means it does not rely on living cells but uses extracts containing the essential elements for protein production.
• The system is fully automated:
  • Add required reagents and kit components.
  • Inject a DNA template (plasmid or PCR product) encoding the protein of interest.
  • ExiProgen then handles the entire process of generating mRNA from the DNA template through IVT and then translating the mRNA into protein.

• Automation: ExiProgen saves time and minimizes human error compared to manual protein synthesis techniques.
• Efficiency: The system is designed for efficient protein production.
• Versatility: Can be used to produce a variety of proteins depending on research needs.
• Speed: Protein synthesis can be completed in a relatively short time compared to traditional methods.
• Scalability: Can produce proteins that are difficult to express in living cells.

Here's a breakdown of protein purification using a histidine tag (His-tag) in a cell-free system:

Cell-Free Protein Synthesis with His-Tagged Protein:

• 1. DNA Construct with His-Tag:
  • You start with a DNA construct encoding the protein of interest. This DNA sequence is often engineered to include a sequence encoding a string of histidine residues (His-tag) at either the N-terminus (beginning) or C-terminus (end) of the protein.
• 2. Cell-Free Protein Synthesis:
  • The cell-free system, containing a cell extract with the necessary machinery for translation, is used to synthesize the protein from the DNA template.
  • During translation, the ribosomes translate the mRNA sequence, incorporating the His-tag along with the desired protein sequence.

Protein Purification using His-Tag:

• 1. Immobilized Metal Affinity Chromatography (IMAC):
  • This is the most common technique for purifying His-tagged proteins. It utilizes a chromatography column containing beads coated with nickel (Ni) ions.
  • Histidine residues in the His-tag have a high affinity for nickel ions.
• 2. Binding and Washing:
  • The cell-free reaction mixture containing the His-tagged protein is passed through the chromatography column.
  • The His-tagged protein specifically binds to the nickel ions on the beads, while other proteins in the mixture flow through the column and are collected as waste.
  • Washing steps are then performed to remove any nonspecifically bound molecules.
• 3. Elution:
  • To elute (release) the purified His-tagged protein, a solution containing a higher concentration of a competing molecule, often imidazole, is introduced.
  • Imidazole competes with the His-tag for binding to the nickel ions, causing the His-tagged protein to detach from the beads and be collected as the purified protein fraction.

Advantages of His-Tag Purification in Cell-Free Systems:

• Specificity: The His-tag provides a specific and efficient way to isolate the protein of interest from the complex cell-free extract.
• Single-Step Purification: IMAC is a relatively simple and fast technique, offering single-step purification from the cell-free reaction mixture.
• Compatibility: Cell-free systems are generally compatible with His-tag purification methods.

Considerations:

• Cleaving the His-Tag (Optional): In some cases, you might want to remove the His-tag after purification. This can be achieved using specific enzymes that cleave the linker sequence between the His-tag and the protein.
• Optimization: Depending on the specific protein and cell-free system used, some optimization of the IMAC conditions (e.g., imidazole concentration for elution) might be necessary.

This is a service that synthesizes the gene for the protein you want to express, expresses the protein using ExiProgen™, purifies it, and then sends it to you.

By adding the gene coding for the protein you want to express to the protein synthesis kit, mounting it on the ExiProgen™ equipment, and operating it according to the manual, you can easily obtain purified proteins.

Standard Gene to Protein Service: This service proceeds with protein synthesis by adding the gene to the protein synthesis kit in the form of a plasmid cloned into an expression vector (pBT7-N-His or pBT7-C-His) after gene synthesis (300 bp to 3000 bp).

Cloning-free Gene to Protein Service: This service proceeds with protein synthesis by adding the gene in the form of a PCR product to a protein synthesis kit after AccuGeneBlock service (300 bp to 1000 bp).

Basically, the provided protein is supplied by dissolving it in the following buffer:

• Typical protein: 50mM Tris-Cl (pH7.6), 100mM NaCl, 1mM DTT, 0.1mM EDTA, 0.05% NaN3, 50% Glycerol
• Protein with disulfide bond: 50mM Tris-Cl (pH7.6), 100mM NaCl, 1mM DTT, 0.05% NaN3, 50% Glycerol

If you wish to use a special buffer, please write it down and send it when you submit your order.

Additional costs and time are required when proteins are supplied by dissolving them in a special buffer.