Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences

Abstract

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.

Ran promotes membrane targeting and stabilization of RhoA to orchestrate ovarian cancer cell invasion

“…Nevertheless, we did not detect endogenous RhoA in the nucleus of TOV-112D or TOV-1946 cells, suggesting that the major signaling responses observed in our study are mainly due to the RhoA translocation to the PM/ruffles. However, in transfected cells, we do on occasion see RhoA signal in the nucleus, although this is most probably due to an artifact associated with overexpression, which has been observed in other mCherry constructs 48 .…”

Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

“…In addition to transfection with the reporter pair, cells received a third plasmid encoding an untagged, wild typic variant of either interaction partner. In contrast to the reporter pair plasmids carrying the HSV-TK promotor, the plasmids used for competition with untagged variants harbored a CMV promotor generally allowing stronger expression of a downstream gene [ 46 ]. An empty vector (EV) transfection was run in parallel and served as negative control.…”

Determination of the number of RAD51 molecules in different human cell lines

“…Reducing ectopic expression level of fusion proteins has been used to minimize impacts on cellular functions [27]. Thus, we created an EGFP-RAD51 fusion using HSV thymidine kinase (TK) instead of CMV promoter sequences yielding moderate protein expression levels and reduced artificial influences on cells (Figure 3(b) lower panels and panel 3D).…”

“…A TK-EGFP plasmid [27] was treated with NheI and AseI restriction enzymes to receive the HSV-TK promotor sequence. Afterward, the HSV-TK promotor sequence was cloned between the NheI and AseI restriction sites in an EGFP-RAD51 vector thereby excluding its CMV promotor sequence.…”