Recombinant Dihydrofolate Reductase (DHFR) Expression, Purification, and Enzyme Activity Assay

These flashcards cover essential terms and concepts related to the recombinant expression and purification of Dihydrofolate Reductase, its enzymatic activity, and the methodologies used in the laboratory.

Recombinant Gene

A gene that has been artificially made by combining parts from different genes. Often created using molecular cloning techniques, allowing for the expression of desired proteins in host organisms.

Plasmid

A small, circular piece of DNA used for gene cloning. Plasmids act as vectors to carry recombinant genes into host cells (e.g., bacteria) for replication and expression, often containing an origin of replication and selectable markers.

Recombinant Protein

A protein that is genetically engineered and produced from recombinant DNA. These proteins are often produced in heterologous systems (e.g., bacteria, yeast, insect, or mammalian cells) for various applications, including research, diagnostics, and therapeutics.

Fusion Construct

A hybrid protein created by linking two or more genes together. One common application is fusing a gene of interest to an affinity tag gene, which facilitates purification or detection of the recombinant protein.

Affinity Tag

A peptide sequence added to a protein to facilitate its purification. Examples include GST tag, His-tag, Strep-tag. These tags bind specifically to a ligand on a chromatography matrix, allowing selective capture of the tagged protein from a complex mixture.

GST Tag

Glutathione S-transferase tag used for protein purification. The GST tag binds specifically to glutathione immobilized on an affinity column, enabling the purification of GST-fusion proteins.

T7 Promoter

A strong bacterial promoter used for controlling gene expression. In expression systems like BL21(DE3) E. coli, the T7 promoter drives high-level transcription of recombinant genes when induced by IPTG, which de-represses T7 RNA polymerase expression.

Dihydrofolate Reductase (DHFR)

An enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate. This reaction is crucial for nucleotide synthesis and cell proliferation. DHFR is a common target for anticancer drugs due to its role in rapidly dividing cells.

NADPH

A coenzyme used in anabolic reactions. Specifically, NADPH is a crucial electron donor for DHFR, reducing dihydrofolate to tetrahydrofolate. It's vital for many biosynthetic pathways.

Substrate

The reactant on which an enzyme acts. For DHFR, its primary substrates are dihydrofolate (DHF) and NADPH.

Folic Acid Analog

A compound resembling folic acid, used in drug therapies. Many DHFR inhibitors are folic acid analogs (e.g., Methotrexate) which competitively bind to the enzyme, blocking its activity.

Methotrexate

A chemotherapy drug that inhibits DHFR. By binding to and inhibiting DHFR, Methotrexate prevents the synthesis of tetrahydrofolate, thereby blocking DNA synthesis and cell division, making it effective against rapidly growing cancer cells.

Enzyme Inhibition

A decrease in enzyme activity due to interference. Can be reversible (competitive, non-competitive, uncompetitive) or irreversible, often crucial for drug development targeting specific enzymes like DHFR.

Metastases

Cancer cells that spread from the original tumor to other parts of the body. Inhibiting enzymes like DHFR is a strategy to combat uncontrolled cell proliferation in primary tumors and prevent the development of metastases.

Therapeutics

Medicines or treatments designed for therapeutic effects. Recombinant proteins are often developed as therapeutics, for example, insulin, growth hormones, or antibodies for various diseases. Enzyme inhibitors also fall into this category.

Therapeutic Proteins

Proteins used as medicinal therapies. These include enzymes, hormones, antibodies, and growth factors, produced using recombinant DNA technology. Recombinant DHFR itself can be a model for studying therapeutic enzyme targets.

Recombinant DHFR

Dihydrofolate reductase that is produced via recombinant techniques. Often expressed in host systems like E. coli for studies on enzyme kinetics, inhibition, and structure, or as a model protein for biopharmaceutical production.

Protein Folding

The process by which a protein assumes its functional shape. Critical for a protein's activity. Improper folding can lead to inactive proteins or aggregates, a major challenge in recombinant protein production.

Post-Translational Modification

Chemical modifications of a protein after translation. These modifications (e.g., glycosylation, phosphorylation, acetylation) can alter a protein's folding, stability, activity, or localization. Bacterial systems often lack the machinery for complex eukaryotic PTMs.

Glycosylation

The addition of sugar molecules to proteins. A common post-translational modification, particularly important for secreted or cell-surface proteins in eukaryotes, affecting their stability, solubility, and immunological properties. Generally absent in bacterial expression systems.

BL21 (DE3) E. coli

A strain of E. coli used for protein expression. This strain contains the gene for T7 RNA polymerase, which is under the control of the lacUV5 promoter, allowing inducible expression of genes cloned downstream of a T7 promoter on a plasmid.

E. coli Growth Medium

Nutrient solution used to cultivate E. coli. Contains carbon sources (e.g., glucose), nitrogen sources, salts, and trace elements. Optimized for bacterial growth, and can be adjusted for high-density fermentation in biomanufacturing.

Cell Culture

Growing cells under controlled conditions. Refers to the broader practice of cultivating cells (bacterial, yeast, mammalian) in vitro for research or industrial production, forming the basis for recombinant protein expression.

Induction

The process of initiating gene expression. In recombinant protein production, induction is often achieved by adding an inducer (e.g., IPTG for the T7 promoter system) to the cell culture, triggering the expression of the target gene.

Cell Lysis

The disruption of cell membranes to release proteins and other cellular components. Can be achieved mechanically (e.g., sonication, French press) or chemically (e.g., detergents, enzymes). The choice depends on the cell type and target protein stability.

Lyse

To break open cells to release their content.

Lysate

The solution containing cellular components after lysis. This crude extract contains the target recombinant protein along with host cell proteins, nucleic acids, and other cellular debris.

Centrifugation

A technique that uses centrifugal force to separate components in a solution. Commonly used after cell lysis to separate insoluble cellular debris and inclusion bodies from the soluble protein fraction, a crucial first step in protein purification.

Soluble Proteins

Proteins that are ready to remain in solution after cell lysis. Recombinant proteins produced in a soluble form are generally easier to purify and are more likely to be correctly folded and active compared to those forming insoluble aggregates (inclusion bodies).

Protein Purification

The process of isolating a specific protein from a complex mixture. Involves a series of chromatographic steps to separate the target protein from contaminants based on properties like size, charge, hydrophobicity, or specific binding affinity.

Affinity Chromatography

A method for purifying proteins based on specific binding interactions. Utilizes a ligand (e.g., glutathione for GST-tagged proteins, metal ions for His-tags, protein A for antibodies) immobilized on a solid matrix. The target protein binds, while contaminants wash through, followed by elution of the purified protein.

Staphylococcus aureus

A bacterium known for producing Protein A, used in affinity purification. Protein A from S. aureus binds specifically to the Fc region of immunoglobulins, making it widely used for the affinity purification of antibodies in therapeutic protein manufacturing.

Immunoglobulins

Antibodies that are produced by the immune system. These Y-shaped proteins are key components of the adaptive immune response. They are a major class of therapeutic proteins, often purified using Protein A affinity chromatography.

Ion Exchange Chromatography

A technique that separates proteins based on their charge. Proteins bind to a charged resin (anion or cation exchanger) and are eluted by changing the salt concentration or pH of the buffer, which neutralizes the charge or competes for binding sites.

Hydrophobic Interaction Chromatography (HIC)

A technique that separates proteins based on their hydrophobicity. Proteins bind to a hydrophobic resin at high salt concentrations and are eluted by decreasing the salt concentration, which reduces hydrophobic interactions.

Size Exclusion Chromatography (SEC)

A method that separates proteins based on their size. Proteins pass through a porous matrix; larger proteins elute first because they are excluded from the pores, while smaller proteins penetrate the pores and elute later. Useful for polishing and buffer exchange steps.

Protein Assay

A method to analyze protein concentration in a sample. Provides a quantitative measure of total protein, essential for downstream applications and for determining purification yield. Examples include Bradford, Lowry, and BCA assays.

Bradford Protein Assay

A colorimetric assay used to measure protein concentration. Based on the binding of Coomassie Brilliant Blue dye to proteins, resulting in a color shift from red-brown to blue, which can be measured spectrophotometrically at 595 nm.

UV Spectrophotometer

An instrument used to measure the absorbance of UV light by a sample. Can be used for quantitative protein analysis by measuring absorbance at 280 nm (due to tryptophan and tyrosine residues) or for monitoring the progress of chromatography by detecting protein elution.

SDS-PAGE

A technique for separating proteins based on their size using polyacrylamide gel. Proteins are denatured by Sodium Dodecyl Sulfate (SDS) and migrate through the gel towards the positive electrode. Smaller proteins move faster, allowing separation by molecular weight.

Sodium Dodecyl Sulfate (SDS)

A detergent used to denature proteins for electrophoresis. SDS binds to proteins, imparting a uniform negative charge and unfolding them, ensuring that separation during SDS-PAGE is based solely on their molecular weight.

Coomassie Stain

A dye used to visualize proteins in SDS-PAGE gels. Coomassie Brilliant Blue binds non-specifically to proteins in the gel, making them visible as blue bands, allowing assessment of protein purity and quantity.

Western Blot

An analytical technique used to detect specific proteins in a sample. Involves transferring proteins from an SDS-PAGE gel to a membrane, followed by immunodetection using specific antibodies to identify and quantify the target protein.

Enzyme Activity Assay

A test to determine the functional activity of an enzyme. Measures the rate at which an enzyme converts its substrate into product, often by monitoring changes in absorbance or fluorescence. Essential for ensuring the purified protein is active.

Enzyme Kinetics

The study of rates of enzyme-catalyzed reactions. Involves measuring reaction rates under different conditions (e.g., varying substrate concentrations) to determine kinetic parameters like Km and V{max}, providing insights into enzyme mechanism and inhibitor efficacy.

Concentration

The amount of a substance in a given volume of solution. A critical parameter throughout protein production and purification, influencing protein stability, activity, and downstream applications. Often measured using protein assays or UV spectrophotometry.

Biomanufacturing

The use of biological systems for producing products, such as proteins. Involves large-scale production of therapeutic proteins, vaccines, and other biologics using engineered cells (e.g., E. coli, yeast, mammalian cells) in bioreactors, followed by complex purification processes.