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RECOMBINANT PROTEINS

3rd June, 2024

RECOMBINANT PROTEINS

Source: Hindu

Disclaimer: Copyright infringement not intended.

Context

  • Researchers at the Indian Institute of Science (IISc), Bengaluru, have developed an innovative method for the mass production of recombinant proteins, presenting a safer and more efficient alternative to existing techniques.
  • This advancement addresses the hazards associated with methanol usage in recombinant protein production, proposing the use of monosodium glutamate (MSG) instead.

Details

Traditional Method: Methanol-Induced Production

Recombinant Proteins:

  • Recombinant proteins are produced by genetically modifying bacterial, viral, or mammalian cells.
  • Applications:These proteins are crucial for various medical applications, including vaccine antigens, insulin, and monoclonal antibodies.
  • Yeast (Pichia pastoris/Komagataella phaffii):The most widely used organism for this production.

Methanol-Induced Process:

  • Promoter Activation:The yeast contains a unique promoter (AOX promoter) that is activated by methanol, which codes for the enzyme alcohol oxidase (AOX).
  • Process:
    • The gene coding for the desired protein is inserted next to the AOX promoter in the yeast genome.
    • Yeast cells are grown using glycerol or glucose.
    • Methanol is added to activate the AOX promoter, inducing protein production.

Challenges with Methanol:

  • Safety Risks:Methanol is highly flammable and hazardous.
  • Oxidative Stress:Methanol metabolism produces hydrogen peroxide, which can damage yeast cells and recombinant proteins.

Novel Method: MSG-Induced Production

Monosodium Glutamate (MSG) as an Alternative:

  • Discovery:MSG, a USFDA-approved food additive, can activate a different promoter in the yeast genome.
  • Promoter:This promoter codes for the enzyme phosphoenolpyruvate carboxykinase (PEPCK).
  • Process:
    • The MSG activates the PEPCK promoter, leading to protein production similar to that induced by methanol.
  • Advantages:
    • Safety:MSG is non-flammable and safer to handle than methanol.
    • Efficiency:The process eliminates the oxidative stress caused by methanol.

Applications

  • Biotech Industries:The novel expression system can be utilized for the mass production of valuable proteins.
  • Products:Includes milk and egg proteins, baby food supplements, nutraceuticals, and therapeutic molecules.
  • Scalability:The method has the potential for widespread adoption in industrial settings due to its safety and efficiency.
  • Innovation:Represents a significant advancement in biotechnological processes, offering a more sustainable and safer approach to recombinant protein production.

About Recombinant Proteins

  • Recombinant proteinsare proteins encoded by genes that have been cloned in a system that supports the expression of the gene and the translation of messenger RNA.
  • This allows for the production of large quantities of a protein that is otherwise difficult to obtain.
  • Recombinant protein technology is pivotal in biochemistry, molecular biology, and medicine.

Production of Recombinant Proteins

Gene Cloning:

  • Isolation of Gene:The DNA sequence encoding the protein of interest is identified and isolated.
  • Vector Selection:The gene is inserted into a plasmid or other suitable vector that will carry the gene into a host cell.
  • Transformation:The vector is introduced into host cells (bacteria, yeast, insect, or mammalian cells).

Expression Systems:

  • Prokaryotic Systems (e.g., E. coli):Simple, fast, and cost-effective. Suitable for non-glycosylated proteins.
  • Eukaryotic Systems:
    • Yeast (e.g., Saccharomyces cerevisiae):Capable of post-translational modifications.
    • Insect Cells (e.g., Baculovirus system):High yield and proper folding for complex proteins.
    • Mammalian Cells (e.g., CHO cells):Best for producing complex proteins with post-translational modifications similar to human proteins.

Protein Purification:

  • Cell Lysis:Release of protein from host cells.
  • Chromatography Techniques:
    • Affinity Chromatography:Uses a specific ligand to purify the target protein.
    • Ion Exchange Chromatography:Separates proteins based on charge.
    • Size Exclusion Chromatography:Separates proteins based on size.

Protein Characterization:

  • SDS-PAGE:Determines the molecular weight of the protein.
  • Western Blot:Confirms the presence of the protein using specific antibodies.
  • Mass Spectrometry:Provides detailed protein mass and structural information.
  • Activity Assays:Assess the functional activity of the protein.

Applications of Recombinant Proteins

Therapeutics:

  • Insulin:Recombinant human insulin for diabetes management.
  • Erythropoietin:Used to treat anemia.
  • Monoclonal Antibodies:Targeted therapies for cancer and autoimmune diseases.

Vaccines:

  • Hepatitis B Vaccine:Produced using yeast expression systems.
  • HPV Vaccine:Utilizes recombinant technology to produce virus-like particles.

Diagnostics:

  • Enzymes:Used in diagnostic assays (e.g., ELISA).
  • Antigens:Used to detect the presence of antibodies in blood samples.

Advantages

  • High Yield:Large quantities of protein can be produced.
  • Purity:Proteins can be purified to a high degree.
  • Consistency:Batch-to-batch consistency in protein production.

Challenges

  • Post-translational Modifications:Prokaryotic systems cannot perform all modifications.
  • Solubility Issues:Some recombinant proteins form insoluble aggregates.
  • Cost:Production in eukaryotic systems can be expensive.

Sources:

Hindu

PRACTICE QUESTION

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