Biotechnology Study Notes: Proteins as Products

Introduction to Biotechnology Chapter 4: Proteins as Products

4.1 Proteins as Biotechnology Products

  • Definition of Proteins: Large molecules essential for the structure, function, and regulation of living cells.

  • NIH Protein Structure Initiative (2000):

    • Objective: Identify human protein structures.

    • 2010: Adoption of high-throughput structure determination to tackle various biological and biomedical challenges.

    • Goals: Model unknown protein structures by comparing against data stored in databases.

    • Achievements:

      • More than 1,200 superfamilies of proteins identified.

      • Solid understanding of the correlation between an amino acid sequence and its structure.

      • Public database contains over 33,000 protein sequences related to the human proteome.

  • Historical Use of Proteins in Industry:

    • Long-established uses in various manufacturing processes:

    • Beer brewing

    • Winemaking

    • Cheese production

  • Recombinant DNA Technology:

    • Enabled on-demand production of specific proteins:

    • Examples include enzymes, hormones, antibodies.

  • Industrial Applications of Enzymes (Table 4.1):

    Enzyme

    Application

    Amylases

    Digest starch in fermentation and processing

    Proteases

    Used in detergents, meat/leather processing, cheese, brewing, baking, digestive aids

    Lipases

    Digest lipids in dairy and vegetable oil

    Pectinases

    Used in fruit juice processing

    Lactases

    Digest milk sugar

    Glucose isomerase

    Produces high-fructose syrups

    Cellulases/Hemicellulases

    Used for animal feeds, fruit juices

    Penicillin acylase

    Produces penicillin

    Alpha-galactosidase

    Treatment for lysosomal storage disease

    Alpha-L-iduronidase

    Treatment for lysosomal storage disease

  • Biotech Drugs and Medical Applications:

    • Produced through microbial fermentation or mammalian cell cultures.

    • Complex, time-consuming production process:

    • Once method is established, proteins can be generated in large batches using bioreactors with transformed host cells that harbor therapeutic genes.

    • Key parameters in culture conditions: temperature, oxygen, acidity, and other factors.

    • Proteins are isolated and rigorously tested through purification.

  • Regulatory Compliance:

    • All stages must comply with FDA regulations.

    • Hypothetical scenario for patients taking Growth Hormone (GH):

    • Implications of improper purification if GH is cloned in bacteria.

  • Protein-Based Pharmaceutical Products (Table 4.2):

    Protein

    Application

    Erythropoietins

    Treatment of anemia

    Interleukins

    Cancer, AIDS, bone marrow suppression

    Monoclonal antibodies

    Used in cancer treatment and diagnostics

    Interferons

    Treatment of cancer, allergies, asthma, arthritis, infectious diseases

    Colony-stimulating factors

    Treatment for cancer, low blood count, adjuvant chemotherapy, AIDS

    Blood clotting factors

    Treatment for hemophilia and related disorders

    Human growth factor

    Treats growth deficiency in children

    Epidermal growth factor

    Wound healing and cancer treatment

    Insulin

    Diabetes management

    Insulin-like growth factor

    Additional diabetes treatment

    Tissue plasminogen activator

    Post-heart attack and stroke treatment

    Tumor necrosis factor

    Cancer therapy

    Vaccines

    Hepatitis B, malaria, herpes prevention

  • Therapeutic Proteins: Considered biotech drugs, primarily produced via microbial fermentation or mammalian cell culture.

    • Over 400 biotechnology medicines in development, mostly comprising proteins.

    • Patient responses to medications are unique, influenced by genetic factors.

    • Biomarkers: Utilized to predict drug efficacy for specific patient profiles.

  • Examples of Biomarker Applications:

    • Identification of protein level variations in pancreatic cancer patients compared to healthy individuals, showing over 90% accuracy in early detection.

  • Innovations in Drug Production:

    • Development of smart bandages that produce antibiotic proteins in response to infections.

    • Research at MIT led to the creation of micro-pellets that autonomously produce proteins for immediate medical use.

  • Bioremediation:

    • Proteins aid in cleaning up harmful pollutants:

    • Organic waste digesting enzymes and sticky proteins like metallothioneins that encapsulate heavy metals to stabilize pollutants without breakdown.

4.2 Protein Structures

  • General Characteristics of Proteins:

    • Comprised of amino acid chains.

    • Exhibit specific molecular weights and electrical charges.

    • Interaction types:

      • Hydrophilic: Water-attracting.

      • Hydrophobic: Water-repelling.

  • Structural Arrangements: Four levels

    • Primary Structure: Sequence of amino acids linked together.

    • Secondary Structure: Folding or twisting of amino acid chains due to hydrogen bonds, forming shapes like:

    • Alpha Helix: Right-handed spiral stabilized by hydrogen bonds between amino acids.

    • Beta Sheet: Parallel or anti-parallel chains linked via hydrogen bonds.

  • Tertiary Structure: 3D polypeptides formed through cross-linking of secondary structures, encapsulating the protein's functional properties.

  • Quaternary Structure: 3D complexes of multiple polypeptides.

  • Protein-Protein Interaction Map:

    • Discovery of molecular machines composed of numerous proteins (e.g., 'Commander' complex) that have implications for understanding human disorders.

  • Importance of Proper Folding:

    • Protein functionality relies on correct folding; misfolded proteins are linked to serious diseases (e.g., Alzheimer’s, cystic fibrosis). Understanding folding processes is vital in biotechnology.

  • Post-Translational Modifications (PTMs):

    • Over 100 different PTMs known to occur within eukaryotic cells, affecting protein activity such as solubility and lifespan inside organisms.

    • Glycosylation: Addition of carbohydrate units to proteins.

  • Example of Glycoprotein Treatment: Glycoproteins combined with nanoparticles for targeted chemotherapy against B lymphoma cancer cells.

  • Protein Engineering: Directed evolution techniques such as PACE (phage-assisted continuous evolution) accelerate protein development significantly.

  • Prion Proteins and Misfolding Diseases:

    • Discussion of how infectious prions induce harmful structural folds leading to diseases like CJD.

4.3 Protein Production

  • Challenges in Protein Production:

    • Proteins are complex and sensitive to environmental conditions.

    • Two phases in protein production:

    • Upstream Processing: Involves actual protein expression within cells.

    • Downstream Processing: Purification and functional verification of the protein.

  • Upstream Processing Options:

    • Bacteria (E. coli):

    • Advantages: Rapid growth, large-scale production potential, well-studied genetics.

      • Method of gene induction to express proteins.

    • Common issues: Formation of inclusion bodies requiring further processing.

  • Protein Expression from Other Organisms:

    • Fungi: Capable of post-translational modifications needed for proper protein folding (Table 4.4).

    • Variety of proteins sourced from fungi (e.g., interferon, lactoferrin).

    • Plants: Can be genetically modified to produce foreign proteins, although traditional glycosylation processes differ.

    • Mammalian Cultures: Best for human proteins though complex and slower growth with contamination risks.

    • Insect Systems: Utilize baculoviruses for mammalian protein expression, though differing PTMs can occur.

  • Downstream Processing:

    • Steps for purification start with cellular extraction and lysis of cells, followed by stabilization of proteins with inhibitors.

    • Various methods for protein separation:

    • Chromatography: Techniques based on size, charge, and binding affinity are employed for separating proteins, including size exclusion (gel filtration), ion exchange, and affinity chromatography.

    • Dialysis and Filtration Methods: Employed post-extraction to refine protein purity effectively.

  • Verification of Purity and Activity:

    • SDS-PAGE: Technique for visualizing protein size and purity based on molecular weight.

    • ELISA: Specific detection of proteins through antibody binding, used to quantify presence and amount.

  • Preservation Techniques:

    • Methods like lyophilization maintain structural integrity for long-term preservation.

4.4 Proteomics

  • Definition of Proteomics: A discipline aimed at understanding the relationship between protein expression and diseases by comparing proteomes under varying health conditions.

  • Techniques Used:

    • Two-dimensional gel electrophoresis followed by mass spectrometry for protein identification.

    • Protein microarrays for detecting and studying protein-protein interactions.

  • Applications in Diagnostics and Drug Development: Focus on protein markers for disease progression and targeted therapies.

  • Progress in Protein Interaction Discoveries: Advances in functional protein microarrays have broadened research into various protein interactions beyond simple binding studies, aiding in novel therapeutic approaches.