Coexpression of DNA Fragmentation Factor Subunits in E.coli by Two Incompatible Plasmids

Area of Science:

• Molecular Biology
• Protein Expression
• Apoptosis Research

Background:

• The human DNA fragmentation factor (DFF) is crucial for apoptosis, mediating DNA fragmentation and chromatin condensation.
• DFF functions as a heterodimer of DFF40 (caspase-activated nuclease) and DFF45 (inhibitory subunit).
• Efficient expression of functional DFF40 requires coexpression with DFF45.

Purpose of the Study:

• To develop a novel method for coexpressing DFF45 and DFF40 in E. coli.
• To optimize the expression levels of both DFF subunits.
• To assess the stability of coexpressed plasmids in E. coli.

Main Methods:

• Amplification of human DFF45 and DFF40 coding regions via RT-PCR.
• Cloning into bacterial expression vectors (pET-28a and pET-21a) conferring kanamycin and ampicillin resistance, respectively.
• Co-transformation of E. coli BL21(DE3) with both recombinant plasmids and induction with IPTG.

Main Results:

• Individual expression of DFF45 and DFF40 achieved significant yields (56% and 22% of total protein).
• Coexpression of DFF45 and DFF40 resulted in efficient production (30% and 17% of total protein).
• Co-transformed E. coli maintained both incompatible plasmids, with >75% resistance to both antibiotics after 14 hours culture.

Conclusions:

• A novel and effective method for coexpressing proteins using two incompatible plasmids was established.
• This method allows for the stable coexpression of DFF45 and DFF40 in E. coli.
• The developed system facilitates further investigation into the function and stability of the DFF complex.