In cell and molecular biology, reporter proteins are commonly used for labeling cell or tissue structures. Reporter proteins are often fluorochromes, which are characterized by the appearance of fluorescence. When illuminated, the molecule absorbs light of a certain wavelength and then emits light of lower energy, i.e. longer wavelengths. To label cell structures with reporter proteins, one usually needs to introduce their genetic record into the organism. The excited red fluorescent protein (RFP) fluoresces red-orange, with a maximum excitation at 558 nm and an emission maximum at 583 nm. Due to the deeper penetration of red light through tissues, red fluorescent proteins are used in the microscopy of thicker samples.
As part of the thesis, we used RFP as a reporter protein and attached the immunoglobulin binding peptide min19Fc_Q6D, with the amino acid sequence GSYWYDVWF, to its N-terminus. Peptides could become an alternative to the bacterial immunoglobulin-binding proteins used as specific ligands for antibodies (e.g., in affinity chromatography to purify monoclonal antibodies from the process fluid), as they have many advantages. We chose the peptide ligand min19Fc_6QD as it displays affinity to the Fc region of immunoglobulins G, to form the fusion protein min19Fc_Q6D-RFP. We were able to monitor its expression due to the reporter protein. We attempted to evaluate its binding to IgGs by fluorescence-linked immunosorbent assay (FLISA).
The gene for the fusion protein was constructed by polymerase chain reaction and inserted into the expression vector. We successfully expressed the recombinant protein in Escherichia coli NiCo21 cells, and purified it by immobilized metal affinity and size exclusion chromatography. We tried to evaluate its binding to the Fc region of IgG antibodies, however, no interaction could be detected. There are several possible explanations for that: i) RFP might sterically hinder the min19Fc_Q6D peptide:antibody interaction; ii) the interaction is thermodynamically disfavoured due to high entropic cost upon binding; and/or iii) the FLISA assay is not sensitive enough to detect binding as the signal is only produced on a molecular monolayer at the bottom of microtiter plate wells.