ecDHFR Plasmids

ecDHFR (Escherichia coli Dihydrofolate Reductase) plasmids are genetic tools used to study the expression, activity, and applications of the DHFR enzyme derived from E. coli. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is essential for nucleotide biosynthesis and cell survival. The bacterial DHFR is a model enzyme often used in protein folding studies, drug screening, and synthetic biology due to its well-characterized structure and function.

Content of ecDHFR Plasmids

• ecDHFR Coding Sequence:
  • Encodes the full-length ecDHFR protein, which is commonly used for structural and functional analysis.
  • May include mutants with altered activity for specific experimental purposes.
• Promoter:
  • Constitutive Promoters: (e.g., T7, lac, or tac) for high expression in bacterial systems.
  • Inducible Promoters: (e.g., IPTG-inducible lac promoter) for controlled expression.
• Tagging Sequences:
  • Fusion tags (e.g., His6, FLAG, or GFP) for purification, tracking, or visualization of ecDHFR.
• Selectable Markers:
  • Antibiotic resistance genes (e.g., ampicillin or kanamycin) for plasmid maintenance in E. coli.
• Replication Origin (Ori):
  • Ensures plasmid replication in bacterial cells (e.g., ColE1 ori).

Applications of ecDHFR Plasmids

• Protein Folding and Stability Studies:
  • ecDHFR is used as a model to study protein folding pathways and the effects of mutations on protein stability.
• Drug Screening:
  • The enzyme serves as a target for screening inhibitors, including antifolates such as trimethoprim and methotrexate.
• Synthetic Biology:
  • Utilized as a selectable marker or reporter in synthetic circuits due to its essential role in folate metabolism.
• Fusion Tag Applications:
  • ecDHFR can be used as a fusion tag for protein expression and solubility enhancement.
• Structural Biology:
  • Crystallographic and biophysical studies leverage ecDHFR as a model enzyme for understanding enzyme-substrate interactions.

ecDHFR plasmids are versatile tools for studying enzyme function, protein folding, and metabolic engineering. Their applications range from fundamental research in structural biology to practical uses in drug screening and synthetic biology. With their ease of use and adaptability, ecDHFR plasmids remain a key resource in biochemistry and molecular biology.