Theoretical basis
Green fluorescent protein: GFP
GFP is a fluorescent protein isolated from the jellyfish Aequorea victoria. It is one of the most widely studied and used proteins in biochemistry and cell biology. It has been established as a marker for gene expression and protein targeting in living cells and organisms. It is 238 amino acids long with a molecular weight of 26.5 kDa. Inside the protein a self-assembled fluorophore is located which leads to its fluorescence properties. The wild type Aequorea protein has a major excitation peak at 395 nm and a minor peak at 475 nm. In aqueous solution (pH= 7.0), excitation at 395 nm leads to an emission peak at 508 nm, whereas excitation at 475 nm causes a maximum emission at 503 nm (visible green light) [4]. In our project, it is used as a fusion protein to provide the ability to detect it by exciting with a wave length of 395 nm.
Besides GFP, which emits a visible green light, there are other versions of fluorescent proteins like the red fluorescent protein (RFP), able to emit all kinds of colors. They are mostly used to label more than one protein of interest and compare their localization and amount in vivo.
Streptavidin
Streptavidin is a tetrameric protein with a molecular weight of 15 kDa for each subunit. It was isolated from the actinobacterium Streptomyces avidinii and is homologous to avidin. Both proteins can bind up to four molecules of biotin and their derivatives with high affinity resulting in the high dissociation constant Kd = 10-15 M. This leads to its widespread use in diagnostic assays and protein tags that require formation of an irreversible and specific linkage between biological macromolecules [7].
In our project, streptavidin is used as a tag to provide the ability to immobilize the target protein on a biotinylated surface.
Fusion Proteins
Fusion proteins have been developed as a class of novel biomolecules with multi-functional properties. By successfully genetically fusing two or more proteins together, the product will have the desired properties from each component. The successful construction of a fusion protein requires the component proteins and suitable linkers. They were inspired by naturally-occurring multi-domain proteins with different subunits that are covalently linked together [6]. Fusion proteins can contain the whole native protein sequences or just parts of it dependent on the desired function and size of the resulting product protein.
In the design process of recombinant fusion proteins, linkers are very important, as they can increase the stability, bioactivity, and expression yield of the fusion protein. Additionally, a direct fusion of functional domains without a linker can lead to misfolding and other undesirable properties. Linker sequences can be received from natural multi-domain proteins or be rationally designed depending on the desired characteristics. They are mainly classified in three different groups: flexible linkers, rigid linkers, and cleavable linkers (see figure X). Furthermore they can be classified as small (approximately five amino acids), medium (approximately ten) or large linkers (approximately 20 to 28 amino acids) [6].
In our project, we used a rationally designed medium-sized gly-gly-ser-linker with a length of eleven amino acids. These small amino acids will allow a good expose of the 2-NPA to the UV-light while the glycines mediate the flexibility and the serines improve the solubility of the fusion protein and prevent a hydrophobic collapse.
Light-induced elution
As an application, we wanted to use 2-NPA in a new purification system for recombinant proteins, similar to affinity chromatography and inspired by a paper form Peters et al. [3]. They cleaved a short model peptide containing 2-NPA to show the ability of the ncAA to induce a cleavage of the protein backbone after irradiation with light with a wavelength of 365 nm. We thought about this model peptide as a linker between the target and the binding protein to establish a new light-induced elution system (Figure X).
We use a recombinant fusion protein with streptavidin as binding unit and a medium gly-gly-ser-linker with 2-NPA connecting the streptavidin with a target protein. As a proof of concept, we used GFP as target protein because of its easy optical detection.
We hope that the fusion protein in unfiltered cell lysate will bind strong and specifically to the purification column with biotinylated glass slides, so that the other proteins and cell fragments can be easily washed away. We then want to irradiate the slides with light of 395 nm wave length to detect the GFP and prove the binding efficiency of the streptavidin and the functionality of the selected linker. Afterwards, we want to irradiate the column with UV-light of 365 nm wave length to induce the photocleavage of the 2-NPA. In the following elution step the GFP will be eluted while other proteins that were bound unspecific to the biotinylated surface should not be effected by the irradiation and retain on the column. The elution of the GFP can then also be detected as well as the fluorescence of the eluate.
After using the purification column it should be easily regenerated by simply washing it with SDS-solution. The SDS will denaturate the streptavidin with the linker and the other proteins bound to the column so that they will lose their binding affinity to the biotin and be washed off the glass slides. The biotin itself should not be influenced by the SDS-solution so that the glass slides will still be usable for many purification steps.
To implement all this, we started the development of a purification column, containing the needed biotinylated surfaces and an LED-panel that is able to radiate the needed UV-light with a wave length of 365 nm.
