Recombinant Protein Overexpression and Purification

Vocabulary flashcards covering key concepts, expression vectors, host strain genetics, induction mechanisms, purification tags, and challenges in recombinant protein overexpression.

Expression Vector (Plasmid)

A genetically engineered circular DNA molecule containing key regulatory elements, such as the phage T7 promoter, LacO operator site, open reading frame (ORF), origin of replication (ori), antibiotic resistance marker, and $lacI$ repressor gene, used to drive inducible target protein expression in host bacteria.

Isopropyl Thiogalactoside (IPTG)

A non-hydrolyzable chemical analog of lactose that binds the lac repressor to induce transcription from the $lac$ operon promoter; because E. coli cannot metabolize it, its concentration remains constant during expression experiments.

E. coli BL21 (DE3)

A fast-growing, non-pathogenic bacterial host strain engineered to lack major intracellular proteases and carrying the DE3 lysogen, which encodes T7 RNA polymerase under the control of the $lac$ promoter for T7-based protein overexpression.

Bacterial Growth Curve

A plot of cell viability over time in batch culture comprising four distinct growth kinetics stages: lag phase, exponential (log) growth phase, stationary phase, and death phase.

Dihydrofolate Reductase (DHFR)

An essential enzyme involved in 1-carbon metabolism that converts 7,8-dihydrofolate to tetrahydrofolate using $\text{NADPH} + \text{H}^+$, serving as a target for therapeutic inhibitors such as Methotrexate and Trimethoprim.

Western Blot

An analytical technique used to identify a specific target protein within a complex mixture by separating proteins via SDS-PAGE, transferring them to a PVDF membrane, blocking non-specific sites, and detecting with target-specific primary and enzyme-conjugated secondary antibodies.

Polyhistidine Tag ($\text{His}_6$ Tag)

An engineered peptide tag consisting of six consecutive histidine residues fused to a protein (e.g., GST-DHFR-$\text{His}_6$) to enable affinity purification via nickel-chelate matrices and targeted detection.

T7 RNA Polymerase Expression System

A high-yield expression control system in which chromosomal T7 RNA polymerase under lac promoter control produces T7 RNA polymerase upon IPTG addition, which in turn transcribes the plasmid-encoded gene of interest under the T7 promoter.

Animal Pancreas Insulin Yield

The historical extraction standard prior to recombinant technology, which required $8\text{,}000\text{ pounds}$ of pancreas glands from $23\text{,}500$ animals to produce just $1\text{ pound}$ of insulin (requiring $56\text{ million}$ animals annually for Eli Lilly).

Post-Translational Modification Challenges

Limitations inherent to bacterial hosts like E. coli that prevent proper eukaryotic folding or function, including the lack of glycosylation machinery and a reducing cytosolic environment that inhibits proper disulfide bond formation.

Bamlanivimab

A recombinantly produced therapeutic antibody supplied as a $700\text{ mg}/20\text{ mL}$ ($35\text{ mg/mL}$) single-dose vial for intravenous infusion under Emergency Use Authorization (EUA).

Fabrazyme (agalsidase beta)

A commercially available recombinant human enzyme product utilized as a biopharmaceutical treatment.