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Recombinant Proteins - Key Concepts and Expression Systems

Key Concepts

  • Recombinant DNA technologies enable production of therapeutic proteins in non-human systems.
  • Eukaryotic expression often required for post-translational modifications (PTMs) and proper folding; some proteins can be produced in prokaryotes but may lack PTMs.
  • Multiple expression platforms exist (bacteria, yeast, insect, mammalian, plants, transgenics); choice depends on PTMs, folding, yield, and cost.
  • Key workflow: design gene construct, clone into expression vector, transform host, grow expressing cells, and purify protein.

Steps to Produce a Recombinant Protein

  • Isolate or generate gene of interest construct.
  • Clone into an appropriate expression vector.
  • Transform into host organism (often bacteria or another system).
  • Grow cells expressing the protein in the chosen system.
  • Purify the expressed protein.

Expression Systems Overview

  • Prokaryotic (Bacteria): fast growth, low cost, high yield; simple proteins; lacks complex PTMs; often requires refolding.
  • Yeast/Insect: some PTMs; better folding for many proteins than bacteria; intermediate cost and speed.
  • Mammalian Cell Culture: authentic PTMs and folding; suitable for complex proteins and antibodies; higher cost and slower growth.
  • Plants: low production costs, scalable, reduced risk of human pathogens; suitable for large-scale needs; potential for oral delivery (edible vaccines).

Prokaryotic Expression (Bacteria)

  • Advantages: fast growth, cost-effective, high yield, relatively simple to scale.
  • Disadvantages: limited or no post-translational modifications; folding problems; some proteins form inclusion bodies.
  • Examples: insulin, human growth hormone (hGH), interferons.
  • Insulin production (case study):
    • Step 1: obtain human insulin cDNA from mRNA via reverse transcription.
    • Step 2: clone gene into expression vectors and transform bacteria.
    • Step 3: grow bacteria expressing insulin chains; insulin A and B chains can be produced separately and then combined.
    • Step 4: purify insulin.
  • Insulin historically: first recombinant human insulin (Humulin) produced in 1978.

Insulin Production Details

  • Insulin is synthesized as a pre-proprotein in pancreas and requires Golgi processing for maturation; bacteria cannot perform this processing, hence A and B chains are produced separately and assembled.
  • Practical steps include expressing A and B chains, purification, and chain joining to form active insulin.

Mammalian Cell Culture for Recombinant Proteins

  • Can perform most PTMs and authentic protein folding; suitable for complex proteins (e.g., EPO, monoclonal antibodies).
  • Common examples: EPO, Factor VIII, tissue plasminogen activator, Enbrel.
  • Erythropoietin (EPO):
    • Post-translational glycosylation is essential for stability and function.
    • Recombinant EPO (rEPO) is produced in CHO cells.
    • Glycosylation increases bloodstream stability, solubility, and proper immune recognition.

Why Make Recombinant EPO?

  • Diseases causing anemia (low red blood cells): chronic renal failure, cancer chemotherapy.
  • rEPO can restore red blood cell levels and improve oxygen transport.

rEPO Details and Detection

  • rEPO is glycosylated differently from natural hEPO due to production in CHO cells; this affects charge and mobility.
  • Detection in athletes: isoelectric focusing (IEF) separates proteins by isoelectric point; different glycosylation patterns cause different pI, enabling discrimination between hEPO and rEPO when visualized with specific antibodies.

Plant-Based Expression Systems

  • Advantages: low cost, scalable, no need for expensive bioreactors, reduced risk of human pathogens, potential for long-term stability in dried tissues, and possible oral delivery.
  • Edible vaccines: plants engineered to express antigens in edible tissues (potatoes, bananas, rice, carrots);
    • Aims for oral immunisation against diseases like measles, cholera, and hepatitis B/C.

Comparison of Expression Systems (Key Points)

  • Growth rate: fastest in bacteria; slower in plants and mammalian systems.
  • Cost: lowest in bacteria; higher in mammalian; plant-based typically low to moderate cost.
  • Protein yield: high in bacteria; moderate to high with optimization in plants/mammalian; variable in some plant systems.
  • PTMs (glycosylation): minimal in bacteria; full in mammalian; partial or distinct in plants.
  • Folding and disulfide bonds: challenging in bacteria; efficient in mammalian; plant systems variable.
  • Secretion: often intracellular in bacteria; often secreted in mammalian; can be apoplastic or secreted in plants.
  • Scalability: highly scalable for bacteria and plants; mammalian systems are scalable but expensive.

Examples: Insulin, EPO, Edible Vaccines

  • Insulin: bacterial production of A and B chains; assembly into active insulin; 1978 Humulin milestone.
  • EPO: rEPO requires mammalian cells for proper glycosylation; used for anemia therapy; detection via IEF to distinguish from natural EPO.
  • Edible vaccines: plant-based expression of antigens for oral immunisation; potential future public health impact.

Key Concepts (Summary)

  • Recombinant DNA enables safe production of human proteins in non-human systems.
  • PTMs are essential for many protein activities; eukaryotic systems are often necessary.
  • A variety of expression systems exist; choice depends on PTMs, folding, yield, and cost.

Practice Questions (Review)

  • What are the key steps to produce a recombinant protein?
  • Why can recombinant insulin be produced in bacteria, and what constraints exist?
  • What are the pros and cons of using a prokaryotic system for human proteins?
  • What are the pros and cons of using a eukaryotic system for human proteins?
  • If EPO were produced in a bacterial system, would it be active in humans?
  • How can rEPO be detected in athletes?
  • What advantages do plant-based systems offer over cell culture for recombinant proteins?