Protein Expression

 Protein expression is the process by which cells synthesize proteins from genetic instructions. It begins with transcription: DNA is copied into messenger RNA (mRNA). During translation, ribosomes decode mRNA to assemble amino acids into polypeptide chains. Post-translational modifications (PTMs)—such as folding, glycosylation, or phosphorylation—then refine the protein into its functional 3D structure. This process occurs naturally in living cells but is harnessed by VLI to produce proteins for research, diagnostics, and therapeutics. 

 Prokaryotes (e.g., Escherichia coli) offer a simple, cost-effective platform for protein production. Target genes are inserted into bacterial plasmids and introduced into host cells. Expression is induced (e.g., via IPTG or temperature shift), leveraging bacterial transcription/translation machinery.

• Advantages: Rapid growth (protein in 24–48 hours), high yields, low cost.
• Limitations:
  • Cannot perform complex PTMs (e.g., mammalian glycosylation).
  • Prone to forming insoluble inclusion bodies (misfolded aggregates).
• Example use: Expressing cytosolic enzymes, non-glycosylated antigens, or protein fragments.

 Eukaryotic systems (yeast, insect, or mammalian cells) support complex proteins requiring sophisticated folding and PTMs.

• Yeast (e.g., Pichia pastoris):
  • Performs basic glycosylation; ideal for secreted proteins.
  • Fast growth, scalable fermentation.
• Insect cells (e.g., baculovirus/Sf9 system):
  • Near-native PTMs; excels for membrane receptors or multi-subunit complexes.
• Mammalian cells (e.g., CHO, HEK293):
  • Gold standard for human-like glycosylation/pharmaceutical proteins.
  • Essential for antibodies, cytokines, and receptors.
• Limitation: Higher cost, longer timelines (weeks vs. days).

  Tags are engineered onto proteins to streamline isolation, solubility and detection:

Polyhistidine (His-tag):

• 6–10 histidine residues bind nickel/cobalt chromatography resins.
• Small size, minimal impact on protein function; works in denaturing conditions.

Glutathione-S-transferase (GST):

• Binds glutathione beads; enhances solubility of fusion partners.
• Removed by protease cleavage (e.g., thrombin, PreScission).

FLAG/HA Epitope Tags:

• Short peptides for antibody-based purification or detection.
• High specificity; minimal interference with protein activity.