Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes...

Area of Science:

• Molecular Biology
• Biotechnology
• Cell Biology

Background:

• Overexpression of fluorescent fusion proteins (FFPs) can cause experimental artifacts and interfere with sensitive microscopy techniques like fluorescence correlation spectroscopy (FCS).
• The strong CMV promoter commonly used for recombinant DNA technologies may lead to excessively high protein expression levels.

Purpose of the Study:

• To identify and characterize alternative promoters for reduced recombinant protein expression.
• To develop novel promoter constructs suitable for sensitive single-molecule and FCS-based biological studies.
• To investigate the regulatory elements within the herpes simplex virus thymidine kinase (TK) gene promoter.

Main Methods:

• Construction and analysis of deletion mutants of the herpes simplex virus thymidine kinase (TK) gene promoter.
• Integration of TK promoter constructs into the Gateway® system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and other FFPs.
• Evaluation of promoter activity and protein expression levels in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cell lines.

Main Results:

• Two TK promoter constructs, TK2ST and TKTSC, demonstrated optimal low expression levels suitable for FCS studies across four different cell lines.
• Promoter constructs with deletions in the TK gene 5'-untranslated region (5'-UTR) exhibited significantly higher protein expression compared to those without deletions.
• Identification of a potential negative regulatory element within the TK gene 5'-UTR.

Conclusions:

• The engineered TK promoter variants provide a valuable tool for modulating FFP expression to avoid artifacts in biological experiments.
• The TK2ST and TKTSC promoters are well-suited for applications requiring precise control over protein expression, such as FCS and single-molecule techniques.
• The TK gene 5'-UTR contains a negative regulatory element that influences protein expression levels.