Expression and purification of codon-optimized cre recombinase in E. coli

Area of Science:

• Biotechnology
• Molecular Biology
• Genetic Engineering

Background:

• Antibiotic resistance genes in genetically modified bacteria pose regulatory challenges for therapeutic protein production.
• Selectable marker gene removal is crucial for optimizing plasmid propagation and ensuring product safety.
• The Cre/loxP recombination system is a widely utilized method for efficient gene excision.

Purpose of the Study:

• To overexpress and purify codon-optimized Cre recombinase (Cre) for selectable marker gene removal.
• To assess the activity of N-terminal His6-tagged Cre recombinase in site-specific recombination.
• To evaluate the utility of different expression vector systems for Cre recombinase production.

Main Methods:

• Codon-optimized cre gene was overexpressed using pColdIV and pET28a(+) vector systems.
• Immobilized metal affinity chromatography (IMAC) was employed for His6-Cre recombinase purification.
• Site-specific recombination activity was tested using a linearized pLox2+ vector containing two loxP sites.

Main Results:

• His6-Cre recombinase was purified with approximately 26-fold enrichment.
• The purified enzyme demonstrated successful site-specific recombination, excising a DNA fragment containing the marker gene.
• Recombination resulted in a re-circularized plasmid and a smaller DNA fragment with the recombined loxP site.

Conclusions:

• N-terminal His6-tagged Cre recombinase can be effectively overexpressed and purified using common expression vectors.
• The purified Cre recombinase exhibits robust enzymatic activity for site-specific DNA recombination.
• This system offers a viable strategy for removing antibiotic resistance genes in biotechnological applications.