Screening for unknown mutations by a bioluminescent protein truncation test with homogeneous detection

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Protein truncation test (PTT) is crucial for identifying mutations leading to premature mRNA translation termination.
• Conventional PTT requires multiple steps including separation and purification of polypeptides, which can be time-consuming.

Purpose of the Study:

• To develop a rapid and sensitive bioluminescent protein truncation test (PTT).
• To eliminate the need for polypeptide separation and purification in PTT.

Main Methods:

• A DNA fragment encoding apoaequorin was fused in-frame to the interrogated sequence.
• The fusion product underwent in vitro coupled transcription and translation in the presence of coelenterazine.
• Detection of a functional chimeric protein was achieved via Ca(2+)-triggered bioluminescence.

Main Results:

• The bioluminescent PTT detected nascent proteins directly in the expression mixture within seconds.
• A wild-type template produced a C-terminal aequorin fusion protein, generating a bioluminescent signal.
• Truncating mutations abolished the production of active photoprotein, resulting in no detectable signal.

Conclusions:

• The developed bioluminescent PTT is a sensitive and rapid method for detecting truncating mutations.
• This assay simplifies the screening process by avoiding protein separation and purification.
• The method was successfully demonstrated for detecting mutations in the APC gene.