RECOMBINANT PROTEINS

Source: Hindu

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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

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