Avoiding the ends: internal epitope tagging of proteins using transposon Tn7

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Peptide tags are crucial for protein purification, detection, and visualization.
• N- or C-terminal fusion of peptide tags can disrupt target protein function.
• Identifying alternative methods for functional tagged fusion proteins is challenging.

Purpose of the Study:

• To develop a method for introducing protein tags internally within a target protein's coding sequence.
• To overcome limitations of N/C-terminal tagging that can impair protein function.
• To create a versatile system for generating functional internally tagged proteins.

Main Methods:

• Utilized in vitro Tn7-transposon mutagenesis for random tag introduction into plasmids.
• Employed Gateway cloning to isolate target gene alleles with internal mutations.
• Designed a Tn7-epitope cassette for precise tag insertion and transposon removal.

Main Results:

• Successfully generated internally epitope-tagged versions of Saccharomyces cerevisiae Dcw1 and Candida glabrata Sir3.
• Demonstrated the ability to insert protein tags at diverse internal sites within the open reading frame (ORF).
• Validated the functionality of internally tagged proteins.

Conclusions:

• The described method provides a robust strategy for internal protein tagging.
• This system facilitates the creation of functional tagged fusion proteins without N/C-terminal disruption.
• The approach is adaptable to various organisms with compatible Gateway expression vectors.