Open AccessOptimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences
Author(s) -
Ali Rizwan,
Ramadurai Sivaramakrishnan,
Barry Frank,
Nasheuer Heinz Peter
Publication year - 2018
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.12432
Subject(s) - green fluorescent protein , microbiology and biotechnology , thymidine kinase , herpes simplex virus , fluorescence microscope , biology , gene , gene expression , recombinant dna , fusion protein , transfection , expression vector , hela , chemistry , cell culture , fluorescence , virus , biochemistry , virology , genetics , physics , quantum mechanics
The modulation of expression levels of fluorescent fusion proteins ( FFP s) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy ( FCS ) and single‐molecule‐based techniques are very sensitive to high expression levels of FFP s. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase ( TK ) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway ® system for ectopic expression of enhanced green fluorescent protein ( eGFP ), monomeric cherry ( mC herry), and FFP s containing these FP s. Two promoter constructs, TK 2 ST and TKTSC , were established, which have optimal low expression levels suitable for FCS studies in U2 OS , HeLa CCL 2, NIH 3T3, and BALB /c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5′‐ UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5′‐UTR.