biotechnology 3 unit 8 study guides

Key Concepts and Terminology

• Protein expression involves the production of a specific protein in a host organism or cell line
• Recombinant DNA technology enables the insertion of a gene of interest into a vector for protein expression
• Vectors are DNA molecules that can replicate independently and carry foreign DNA into host cells
  • Common vectors include plasmids, bacteriophages, and viral vectors
• Host cells are organisms or cell lines used to express the protein of interest
  • Examples include bacteria (E. coli), yeast (S. cerevisiae), and mammalian cell lines (CHO, HEK293)
• Transformation is the process of introducing foreign DNA into a host cell
• Induction refers to the activation of gene expression, often using specific compounds or environmental conditions
• Protein extraction involves the isolation of the expressed protein from the host cell or culture medium
• Purification techniques are used to separate the target protein from other cellular components and contaminants
• Quality control assesses the purity, activity, and stability of the expressed protein

Protein Expression Systems

• Protein expression systems are used to produce recombinant proteins in host cells
• Bacterial expression systems, such as E. coli, are widely used due to their simplicity, rapid growth, and high yields
  • Drawbacks include the lack of post-translational modifications and the formation of inclusion bodies
• Yeast expression systems, like S. cerevisiae and P. pastoris, offer eukaryotic post-translational modifications and secretion of proteins into the culture medium
• Mammalian expression systems, including CHO and HEK293 cells, provide proper folding and complex post-translational modifications
  • Suitable for producing therapeutic proteins and antibodies
• Baculovirus-insect cell expression systems combine the benefits of eukaryotic modifications with high yields
• Cell-free expression systems use cell extracts to produce proteins in vitro, allowing for rapid screening and optimization

Gene Cloning and Vector Design

• Gene cloning involves the insertion of a gene of interest into a vector for expression in a host cell
• The gene of interest is amplified using PCR or synthesized de novo
• Restriction enzymes are used to create compatible ends on the gene and vector for ligation
• Vectors are designed to include essential elements for replication, selection, and expression
  • Origin of replication (ori) allows for vector replication in the host cell
  • Selectable markers, such as antibiotic resistance genes, enable the selection of transformed cells
  • Promoters control the expression of the gene of interest
    • Inducible promoters (T7, lac) allow for controlled expression
    • Constitutive promoters (CMV, SV40) provide continuous expression
• Affinity tags (His-tag, GST-tag) can be added to the protein sequence to facilitate purification
• Codon optimization improves expression by aligning the gene sequence with the host cell's codon usage preferences

Host Cell Selection and Transformation

• Host cell selection depends on the desired protein characteristics, post-translational modifications, and scalability
• Bacterial hosts, like E. coli, are suitable for simple, high-yield protein production
  • Strains include BL21(DE3), Rosetta, and Origami for enhanced expression and folding
• Yeast hosts, such as S. cerevisiae and P. pastoris, offer eukaryotic modifications and secretion
• Mammalian hosts, including CHO and HEK293 cells, provide complex modifications and are used for therapeutic proteins
• Transformation methods introduce the vector into the host cell
  • Chemical transformation uses calcium chloride and heat shock to permeabilize bacterial cells
  • Electroporation applies an electric field to create pores in the cell membrane
  • Transfection methods, like lipofection and calcium phosphate, are used for mammalian cells
• Screening and selection of transformed cells ensure the presence and expression of the gene of interest

Induction and Culture Conditions

• Induction initiates or enhances protein expression in the host cell
• Inducible promoters allow for controlled expression at the desired stage of cell growth
  • IPTG induces expression in the lac operon system by binding to the lac repressor
  • Galactose induces expression in the GAL promoter system by activating the GAL4 transcription factor
• Induction conditions, such as inducer concentration, temperature, and duration, are optimized for each protein and host cell
• Culture conditions impact cell growth, protein expression, and protein quality
  • Media composition provides necessary nutrients and energy sources for cell growth
    • Rich media (LB, YPD) contain complex nutrients, while defined media have specific components
  • Temperature affects cell growth rate, protein folding, and solubility
    • Lower temperatures (16-25°C) can improve soluble protein expression in bacteria
  • pH and aeration influence cell metabolism and protein production
• Fed-batch and continuous culture strategies can enhance protein yields and minimize toxic byproduct accumulation

Protein Extraction and Cell Lysis

• Protein extraction involves the isolation of the expressed protein from the host cell or culture medium
• Cell lysis methods disrupt the cell membrane and release intracellular proteins
  • Mechanical lysis uses physical forces, such as sonication or high-pressure homogenization
  • Enzymatic lysis employs enzymes (lysozyme) to degrade the cell wall
  • Chemical lysis uses detergents (Triton X-100, SDS) or chaotropic agents (urea, guanidine hydrochloride) to solubilize proteins
• Lysis buffer composition is optimized to maintain protein stability and minimize degradation
  • Protease inhibitors prevent unwanted protein degradation by endogenous proteases
  • Reducing agents (DTT, β-mercaptoethanol) break disulfide bonds and maintain protein reduction
• Centrifugation or filtration separates the soluble protein fraction from cell debris
• Secreted proteins can be directly isolated from the culture medium, simplifying the extraction process

Purification Techniques

• Protein purification aims to isolate the target protein from other cellular components and contaminants
• Affinity chromatography exploits the specific binding between the target protein and an immobilized ligand
  • His-tagged proteins bind to immobilized metal ions (Ni-NTA, Co-NTA)
  • GST-tagged proteins interact with glutathione-coupled resins
• Ion exchange chromatography separates proteins based on their surface charge
  • Anion exchange resins (Q, DEAE) bind negatively charged proteins
  • Cation exchange resins (S, CM) bind positively charged proteins
• Size exclusion chromatography (gel filtration) separates proteins based on their molecular size and shape
• Hydrophobic interaction chromatography separates proteins based on their surface hydrophobicity
• Reversed-phase chromatography separates proteins based on their hydrophobicity using a non-polar stationary phase
• Multi-step purification strategies combine different techniques to achieve high purity
• Dialysis or desalting removes small molecules and buffer exchanges the purified protein

Analysis and Quality Control

• Protein analysis and quality control assess the purity, activity, and stability of the expressed protein
• SDS-PAGE and Western blotting evaluate protein purity and confirm the presence of the target protein
  • SDS-PAGE separates proteins based on their molecular weight
  • Western blotting uses antibodies to specifically detect the target protein
• Protein concentration is determined using colorimetric assays (Bradford, BCA) or UV absorbance at 280 nm
• Protein activity assays measure the biological function of the purified protein
  • Enzyme assays monitor substrate conversion or product formation
  • Binding assays assess the interaction between the protein and its ligand or receptor
• Mass spectrometry confirms the protein identity and detects post-translational modifications
• Circular dichroism and fluorescence spectroscopy provide insights into protein folding and stability
• Endotoxin testing ensures the absence of bacterial lipopolysaccharides in the purified protein sample
• Sterility testing verifies the absence of microbial contamination

Applications and Case Studies

• Recombinant protein expression has diverse applications in research, biotechnology, and medicine
• Therapeutic proteins, such as insulin, growth factors, and monoclonal antibodies, are produced using recombinant techniques
  • Insulin production in E. coli revolutionized the treatment of diabetes
  • Monoclonal antibodies, like adalimumab (Humira), are expressed in mammalian cells for the treatment of autoimmune disorders
• Industrial enzymes, including proteases, lipases, and amylases, are expressed in microbial hosts for various applications
  • Proteases are used in detergents, food processing, and leather treatment
  • Amylases are employed in starch processing and biofuel production
• Research applications involve the expression of proteins for structural studies, functional characterization, and interaction analysis
  • Membrane proteins, like G protein-coupled receptors (GPCRs), are expressed in mammalian or insect cells for drug screening and structural biology
  • Fluorescent proteins, such as green fluorescent protein (GFP), are widely used as reporters and fusion tags
• Vaccine development utilizes recombinant protein expression for the production of subunit vaccines and virus-like particles (VLPs)
  • Hepatitis B surface antigen (HBsAg) is expressed in yeast for the production of the hepatitis B vaccine
  • Human papillomavirus (HPV) L1 protein forms VLPs when expressed in insect cells, serving as the basis for the HPV vaccine