Proteins as Biotechnology Products

Overview of Proteins as Biotechnology Products

• Proteins are complex molecules fundamental to biotechnology manufacturing processes. The central challenge in this field is understanding and controlling protein folding during production.

• Key Protein Characteristics:

  • Comprised of chains of amino acids.

  • Possess specific molecular weights.

  • Exhibit electrical charges that facilitate interactions with other molecules.

  • Hydrophilic: "Water-loving" regions.

  • Hydrophobic: "Water-hating" regions.

Protein Structure and Folding

• The functionality of a protein is entirely dependent on its structure and correct folding.

• Misfolding Consequences:

  • If a protein is folded incorrectly, its desired function is lost.

  • Misfolded proteins can be detrimental and are linked to various diseases, including Alzheimer's, cystic fibrosis, mad cow disease, certain forms of cancer, and specific types of heart attacks.

• Historical Context:

  • In 1951, Pauling and Corey described two regular secondary structures: alpha helices and beta sheets.

• Structural Fragility: Protein structures are fragile because the hydrogen bonds maintaining them are easily broken.

Four Levels of Structural Arrangement

• Primary Structure: The specific sequence in which amino acids are linked together.

• Secondary Structure: Occurs when amino acid chains fold or twist at specific points due to hydrogen bonds forming between hydrophobic amino acids. Common shapes include:

  • Alpha Helix: A right-handed spiral stabilized by hydrogen bonds linking an amino acid's nitrogen atom to the oxygen atom of another amino acid.

  • Beta Sheet: Hydrogen bonds link nitrogen and oxygen atoms to form sheets. These can be parallel (chains running in the same direction) or anti-parallel (chains alternating in direction).

• Tertiary Structure: Formed when secondary structures are cross-linked to create three-dimensional polypeptides. This level of structure determines the protein's specific function.

• Quaternary Structure: Unique, globular, three-dimensional complexes composed of several participating polypeptide chains.

Post-Translational Modifications (PTMs)

• Glycosylation: A post-translational modification where carbohydrate units are added to specific locations on proteins. In eukaryotic cells, more than $100$ different post-translational modifications can occur.

• Effects of PTMs on Protein Activity:

  • Increasing protein solubility.

  • Orienting proteins into membranes.

  • Extending the active life of a molecule within an organism.

• Major Types of PTMs:

  1. Phosphorylation

  2. Methylation

  3. Sulfation

  4. Acetylation

  5. Amidation

  6. Hydroxylation

  7. Sumoylation

  8. Nitration

  9. Palmitoylation

  10. Formylation

  11. Glycosylation

  12. Ubiquitination

Medical Applications and Glycoproteins

• Glycoproteins as Cancer Treatment: A new method to target and destroy B lymphoma cancer cells involves glycoproteins.

• Mechanism of Action:

  • Glycoproteins are combined with a nanoparticle loaded with a chemotherapy drug.

  • The nanoparticles target cancer cells with specific receptors for these glycoproteins.

  • This increases the effective dose of the drug at the target site while protecting normal healthy tissues.

• Additional Healthcare Uses:

  • Treatment of damaged corneas using biosynthetic corneas made from synthetically cross-linked recombinant human collagen produced in yeast cells.

  • Screening for disease biomarkers using monoclonal antibodies, such as the Prostate-Specific Antigen ($PSA$) test.

Industrial and Pharmaceutical Applications of Proteins

• Historical Uses: Beer brewing, winemaking, and cheese making.

• Recombinant DNA Technology: Allows for the on-demand production of specific proteins such as enzymes, hormones, and antibodies.

• Industrial Enzymes (Table 4.1):

  • Amylases: Digest starch in fermentation and processing.

  • Proteases: Digest proteins for detergents, meat/leather, cheese, and digestive aids.

  • Lipases: Digest lipids (fats) in dairy and vegetable oil products.

  • Pectinases: Digest enzymes in fruit juice/pulp.

  • Lactases: Digest milk sugar.

  • Glucose Isomerase: Produces high-fructose syrups.

  • Cellulases/Hemicellulases: Produce animal feeds and fruit juices.

  • Penicillin Acylase: Produces penicillin.

• Pharmaceutical Products (Table 4.2):

  • Erythropoietins: Anemia treatment.

  • Interleukins $1, 2, 3, 4$: Cancer, AIDS, and bone marrow suppression treatment.

  • Monoclonal Antibodies: Cancer and rheumatoid arthritis treatment; diagnostics.

  • Interferons (alpha, beta, gamma): Cancer, allergies, asthma, and infectious disease treatment.

  • Blood Clotting Factors: Hemophilia treatment.

  • Human Growth Factor: Growth deficiency in children.

  • Insulin/Insulin-like Growth Factor: Diabetes mellitus treatment.

  • Tissue Plasminogen Factor: Treatment after heart attack or stroke.

  • Vaccines: Vaccination against Hepatitis B, malaria, and herpes.

Protein Engineering

• Directed Molecular Evolution: A method that mimics natural selection to evolve proteins or nucleic acids toward a user-defined goal. The process involves:

  • Gene library creation.

  • Mutagenesis (e.g., mutagenic PCR) to induce random mutations.

  • Screening for fitness differences (activity assays).

  • Isolation and amplification of desired variants.

• Site-Directed Mutagenesis: Introducing specific, predefined alterations in the amino acid sequence.

  • Example: Engineering bacteria and enzymes to tolerate cyanide concentrations that are normally lethal.

Biotech Production and Bioreactors

• Target proteins are produced via microbial or mammalian cell culture in bioreactors.

• Bioreactor: A cell system specifically designed to produce biological molecules.

• Culture Conditions: Computers monitor precise variables including temperature, oxygen levels, and acidity ($pH$).

• Regulation: All stages of the process must comply with $FDA$ regulations.

Upstream Processing: Protein Expression

• Upstream processing includes the actual expression of the protein in the host cell.

• Bacteria ($E. coli$):

  • Advantages: Well-understood fermentation, rapid growth, easy genetic alteration.

  • Disadvantages: Proteins often accumulate in insoluble clumps called inclusion bodies; lack of mammalian-like folding; some proteins remain inactive in humans.

  • Strategy: Genetically engineering fusion proteins where the bacterial protein (enzyme) binds to a substrate for easier purification.

  • Activation: Foreign gene expression is stimulated using promoters (e.g., $IPTG$ binding to the lac repressor) once natural metabolism-related protein synthesis is complete.

• Fungi:

  • Eukaryotic hosts capable of post-translational modifications for proper folding.

  • Used for human interferon, human lactoferrin, and bovine chymosin.

• Plants:

  • Rapid growth and high reproductive rates. Tobacco is a common choice.

  • Disadvantages: Presence of cell walls; glycosylation processes differ slightly from animal cells.

• Mammalian Cell Culture:

  • Challenging due to complex nutritional requirements and slow growth; high contamination risk.

  • Best choice for proteins intended for human use.

• Animal Bioreactor Systems:

  • Used for monoclonal antibody production. Mice are injected with an antigen, and the resulting antibodies are purified.

• Insect Systems:

  • Use $Baculoviruses$ to insert mammalian DNA into insect cells. Typically used for small-scale research.

Downstream Processing: Protein Purification

• Downstream processing involves purification, function verification, and stabilization.

• Step 1: Preparing Extract

  • Intracellular proteins: Require cell lysis (disruption of cell walls via freeze/thaw, detergents, or mechanical methods).

  • Extracellular proteins: Recovered directly from culture medium via pipette.

• Step 2: Stabilization

  • Maintain low temperature and proper $pH$ in buffering solutions.

  • Add protease inhibitors and antimicrobials.

• Step 3: Separation Methods

  • Protein Precipitation: Removing water using salts ($ammonium$ $sulfate$) or solvents ($ethanol$, $acetone$, etc.).

  • Centrifugation: Separating components by high-speed spinning; results in a supernatant and a pellet.

  • Membrane Filtration (Diafiltration): Microfiltration (removes bacteria/precipitates) and Ultrafiltration (separates large from small proteins). Clogging is a common issue.

  • Dialysis: Uses a semi-permeable membrane and the concept of equilibrium to remove salts/additives.

  • Chromatography:

    • Size Exclusion: Porous gel beads act as filters; large proteins elute first because they cannot enter the beads.

    • Ion Exchange: Uses electrostatic charge. Anion exchange resin is positively charged; Cation exchange resin is negatively charged.

    • Affinity: Relies on specific, reversible binding to ligands. Often used for fusion proteins.

    • Hydrophobic Interaction: Sorts proteins based on their repulsion of water.

  • 2-Dimensional Gel Electrophoresis: Separates based on isoelectric point ($pH$ where charge is neutral) followed by $SDS-PAGE$ for molecular weight.

Verification of Protein Purification

• SDS-PAGE (Polyacrylamide Gel Electrophoresis):

  • $SDS$ ($sodium$ $dodecyl$ $sulfate$) detergent is added and the mixture is heated to distribute negative charges evenly.

  • Separation is dependent strictly on size and mass.

  • Proteins are visualized using $Coomassie$ stain.

• Western Blotting:

  • Proteins are transferred from the gel to a nitrocellulose membrane via electric current.

  • Primary antibodies bind to the target protein.

  • Secondary anti-immunoglobulin antibodies (coupled to a reporter) bind the primary antibody.

  • A substrate is added to produce a visible band where the target protein is located.

Questions & Discussion

• Question: Why are proteins so fragile?

• Answer: Because they rely on hydrogen bonds that are easily broken.

• Question: Why might normal chemotherapy not be as effective as glycoprotein treatment?

• Answer: Normal chemotherapy affects healthy tissues as well as cancerous ones, whereas glycoprotein-targeting increases the effective dose specifically at the cancer cell site.

• Question: What additional enzymes studied in Chapter 3 would need to be mass produced for recombinant DNA technology?

• Answer: This requires a review of restriction enzymes, ligases, and polymerases used in gene cloning (context implied from transcript references).

• Question: What is an antibody?

• Answer: Proteins produced in response to antigens (viruses, bacteria) that combine with and neutralize those antigens as part of the immune response.

• Question: Which level of structural arrangement in proteins is used to describe 3-dimensional polypeptides formed when secondary structures are cross-linked?

• Answer: Tertiary Structure.

• Question: What are some of the disadvantages of using E. coli for recombinant protein production?

• Answer: Options include inclusion bodies, lack of correct folding, and inactivity in humans (All of the above based on Table 4.3).

• Question: Which of the following is most likely to occur during upstream processing?

• Answer: Expression of the protein in the cell.

• Question: Which type of downstream processing relies on the ability of most proteins to bind specifically and reversibly to ligands?

• Answer: Affinity chromatography.