Protein Synthesis and Purification Techniques

These flashcards cover essential vocabulary and concepts related to protein synthesis, purification techniques, and related biochemical methods.

Protein Expression

The process by which proteins are synthesized and produced in cells.

Origin of replication

The specific sequence where replication starts on the DNA molecule.

Cloning vector

A plasmid or other vector used to carry and replicate foreign DNA.

Bacterial DNA

The genetic material contained within bacteria.

Uptake by bacterial cell

The process through which foreign DNA is absorbed into a bacterial cell.

Peptide synthesis

The process of constructing peptides from amino acids.

Dialysis

A method for separating small molecules from larger molecules in solution. Take protein, metabolites, salts in solution into a bag with holes so that protein stays in the bag while we change the buffer solution. Dialyze it against buffer of choice.  This technique is commonly used in protein purification to remove low molecular weight impurities.

Column Chromatography

A technique for separating substances based on their differential affinities to a stationary and a mobile phase. The stationary phase is something that the product binds on and mobile phase is something that moves. This method allows for the isolation of compounds by passing a mixture through a column filled with solid adsorbent, facilitating the separation of components as they travel at different rates. (equivalent to TLC)

Ion-exchange chromatography

A method of separating and purifying proteins based on their net charge. Protein has positive charge and stationary phase has negative charge. Positive charge will stick, and as you add more salt, more things will stick. The proteins bind to the resin while negatively charged molecules pass through, allowing for elution based on ionic strength.

Size-exclusion chromatography

A technique that separates molecules based on their size. Gel beads have pores that small molecules can go through but big ones cannot. This allows larger molecules to elute first, followed by smaller ones, effectively fractionating the sample based on molecular size (small ones go through all the pores, get distracted and have a much longer path, come off later. big ones cannot go through the pores so they go around the beads, come off faster)

Affinity chromatography

A method that separates proteins based on their specific interactions with a ligand. This technique involves attaching a ligand to a solid support, allowing the target protein to bind specifically, while other proteins are washed away. Once bound, the target can be eluted by changing the conditions to disrupt the interaction. (Ex: ATP analogs - protein binds to resin thinking it is ATP)

Acrylamide Gel Electrophoresis

A technique used to separate proteins based on their size and charge. Separating protein in electric field, based on charge to mass ratio. Bigger molecules: agarose gel and smaller molecules: acrylamide. Big molecules go to top and take longer, while smaller ones go to bottom straight through pores. This method provides high resolution for protein separation, allowing visualization and analysis after staining.

SDS-PAGE

A specific type of electrophoresis that uses sodium dodecyl sulfate to denature proteins. The way that samples move through the gel is logarithmic, not linear. SDS-PAGE allows separation of proteins based on their molecular weight after denaturation. This technique provides insights into protein purity and subunit composition, enabling accurate comparisons.

Mass Spectrometry

An analytical technique used to measure the mass-to-charge ratio of ions. Protein sample is ionized, flies through the tube, and how many seconds it takes shows the mass.

MALDI-TOF

A time-of-flight mass spectrometry technique that uses matrix-assisted laser desorption/ionization. On a surface, zap with laser into gas phase then ionizes it. Then crystallizes with another molecule that likes to go into the gas phase. Bigger molecules go more slowly. This method is useful for analyzing large biomolecules, including proteins, and allows for rapid identification and characterization based on their mass.

Edman Degradation

A method for sequencing amino acids in proteins by systematically removing amino acids from the amino terminal.

2D electrophoresis

A method that separates proteins based on their isoelectric point and molecular weight. First separate by pH (pI/charge). As protein goes through the gel, it gets to the pI, then after it does not have a charge. It then undergoes second dimension separation based on molecular weight using SDS-PAGE.

Centrifugation

A process that uses centrifugal force to separate particles from a solution based on their size, shape, density, medium viscosity, and rotor speed.