Functional domains in Fok I restriction endonuclease

Area of Science:

• Molecular Biology
• Protein Engineering

Background:

• The Flavobacterium okeanokoites restriction endonuclease (fokIR) gene requires optimization for high-level expression in Escherichia coli.
• Understanding the functional domains of Fok I is crucial for its biotechnological applications.

Purpose of the Study:

• To enhance the expression of the Fok I restriction endonuclease in E. coli.
• To delineate the DNA recognition and cleavage domains of the Fok I enzyme.

Main Methods:

• Polymerase chain reaction (PCR) was used to modify transcriptional and translational signals.
• Ribosome-binding site and retroregulator sequences were altered for optimal expression.
• Protein purification involved phosphocellulose, DEAE-Sephadex, and gel chromatography.
• Trypsin digestion and gel-mobility shift assays were employed to analyze protein domains.

Main Results:

• Fok I expression was increased to 5-8% of total cellular protein in E. coli.
• Purification yielded 50 mg of pure Fok I endonuclease per liter of culture.
• Trypsin digestion revealed distinct fragments: a 58-kDa carboxyl-terminal and an 8-kDa amino-terminal fragment in the absence of DNA.
• In the presence of DNA, Fok I cleaved into 41-kDa amino-terminal and 25-kDa carboxyl-terminal fragments.
• The 41-kDa fragment was identified as the DNA recognition domain, while the 25-kDa fragment served as the cleavage domain.

Conclusions:

• Optimized genetic elements significantly boosted Fok I expression in E. coli.
• The Fok I restriction endonuclease comprises separate, identifiable DNA recognition and cleavage domains.
• The 41-kDa amino-terminal fragment specifically recognizes DNA, and the 25-kDa carboxyl-terminal fragment possesses non-specific cleavage activity.