Proteins as Biotechnology Products

Flashcards covering protein structure, biotechnology production processes, engineering methods, and purification techniques including chromatography and electrophoresis.

Proteins

Complex molecules built of chains of amino acids that have specific molecular weights and electrical charges causing them to interact with other molecules.

Hydrophilic

A term describing molecules that are "water loving."

Hydrophobic

A term describing molecules that are "water hating."

Protein Folding

The process upon which the structure and function of a protein depends; incorrect execution can lead to diseases such as Alzheimer's, cystic fibrosis, and mad cow disease.

Pauling and Corey (1951)

Scientists who described two regular secondary structures: Alpha helices and Beta sheets.

Primary structure

The first level of structural arrangement, defined as the sequence in which amino acids are linked together.

Secondary structure

Occurs when chains of amino acids fold or twist at specific points due to hydrogen bonds between hydrophobic amino acids.

Alpha helix

A secondary structure where amino acids form a right-handed spiral stabilized by hydrogen bonds linking a nitrogen atom to an oxygen atom of another amino acid.

Beta sheet

A secondary structure where hydrogen bonds link nitrogen and oxygen atoms to form parallel or anti-parallel sheets.

Tertiary structure

Three-dimensional polypeptides formed when secondary structures are cross-linked; this level determines the protein's function.

Quaternary structure

Unique, globular, three-dimensional complexes built of several polypeptides.

Glycosylation

A post-translational modification where carbohydrate units are added to specific locations on proteins, which can increase solubility and extend active life.

Bioreactor

A cell system used to produce large batches of biological molecules through precise culture conditions such as temperature, oxygen, and acidity.

Amylases

Enzymes used in industry to digest starch in fermentation and processing.

Proteases

Enzymes used to digest proteins for products like detergents, meat, leather, and cheese.

PSA (Prostate-Specific Antigen) Test

An example of using monoclonal antibodies to identify an early predictor biomarker protein for disease diagnosis.

Directed molecular evolution

A protein engineering method that mimics natural selection to evolve proteins or nucleic acids toward a user-defined goal.

Site directed mutagenesis

A process of introducing specific, predefined alterations in the amino acid sequence of a protein.

Upstream processing

The phase of protein production that includes the actual expression of the protein in the cell.

Downstream processing

The phase of protein production involving the purification of the protein, verification of its function, and preservation.

Inclusion bodies

Insoluble clumps of foreign protein that accumulate in the cytoplasm of $E. coli$ and must be purified.

Fusion protein

A protein produced by genetically engineering $E. coli$ where a desired protein is joined to a bacterial enzyme that can bind to a purification column.

IPTG

A substance that binds to the lac repressor without being degraded, used to activate the bacteria promoter and stimulate foreign gene expression.

Baculoviruses

Viruses that infect insects, used as vehicles to insert mammalian DNA into insect cells for protein production.

Cell lysis

The process of disrupting the cell wall to release intracellular proteins, using methods like freeze/thaw or detergents.

Diafiltration (Membrane filtration)

A size-based separation method utilizing membranes with varying pore sizes to filter out cellular debris.

Dialysis

A purification method relying on equilibrium to remove smaller salts and additives by passing them through a semi-permeable membrane.

Size exclusion chromatography

A separation method using porous gel beads where large proteins flow around the beads and elute first, while small proteins enter the beads and move slower.

Ion exchange chromatography

A method that relies on electrostatic charge to bind proteins to resin beads; includes Anion (positive) and Cation (negative) resins.

Affinity chromatography

A purification method relying on the ability of proteins to bind specifically and reversibly to ligands; often used for fusion proteins.

Hydrophobic interaction chromatography

A method where proteins are sorted based on their repulsion of water and attraction to hydrophobic molecules on column beads.

Isoelectric point

The specific $pH$ at which a protein has a neutral charge.

SDS-PAGE

Polyacrylamide gel electrophoresis where the detergent SDS distributes negative charges evenly along denatured proteins to separate them by size and mass.

Western blotting

A verification method where proteins are transferred from a gel to a nitrocellulose membrane and detected using primary and secondary antibodies.