Introduction to the FRET Test
A fluorophore such as green fluorescent protein (GFP) engages in fluorescence in the visual spectrum. This means it has the ability to absorb a short wavelength photon and emit multiple longer wavelength photons. For example, cyan fluorescent protein (CFP) has an excitation wavelength at 400 nm and emits photons to give a peak at 430 nm; however yellow fluorescent will not be excited at 400 nm and requires an excitation of a longer wavelength which is closer to the emission peak at 530 nm. When two distinct fluorophores are in close proximity (within 10 nm) a resonance energy transfer (FRET) can occur, resulting in a relative change in emission peak magnitudes between fluorophores. For example, excitation at 400 nm of a CFP-YFP pair would give a reduction in the CFP peak and an increase in the YFP peak amplitudes. Due to the strong distance dependence of FRET, an observed energy transfer event is a good indication for the successful association of two proteins.
Methods and Results
With the eventual aim of expressing both CIB1 and CRY2 within a single E. coli cell, we designed a construct for FRET containing both a fusion of CIB1 with CFP and a fusion of CRY2 with YFP. It was hypothesised that after growing the cells in the dark, a fluorescence spectrum with excitation at 400 nm would produce only a CFP peak (as no FRET would occur with the CRY2 and CIB1 domains separate). Then upon induction with blue light, the CRY2 and CIB1 domains would associate, producing a FRET and an observable shift in the peak magnitudes, thus confirming their successful association.
After transforming the construct to BL21 E. coli and culturing with IPTG induction, a very low expression level was observed as determined by the fluorescence of the peaks being comparable to background lysate. Excitation at 400 nm produced a small CFP and YFP peak, and blue light induction for 10 min showed no observable change in the spectrum. Unfortunately the individual fusion proteins could not be separated or purified due to the location of the purification tags.
It was then hypothesised that the expression of both fusions on the single plasmid was resulting in reduced expression levels due to the lengthy recombinant sequences not being supported by the E. coli system. From here we decided to redesign the constructs.
In order to conduct a well-defined FRET assay, we required the fusions to be expressed separately, allowing separate spectra to be acquired for the CFP and YFP as a reference. The purified proteins could then be combined in the dark and their spectra compared before and after blue light induction, allowing measurement of any FRET that may occur. Furthermore, appropriate purification tags were to be included to conduct the assay in the absence of any interfering fluorophores within E. coli. A recent paper discusses the importance of the C-terminus of CRY2 and the N-terminus of CIB1 being free for successful mediation of their associative interaction. Thus to prevent interference from the fused GFP, the fusion protein was designed to be fused to the other terminus of each protein. This gave the following final design for the two separate constructs.
In an attempt to obtain the CFP-CRY2 fusion protein, the transformed cells were then cultured in five different growth media, including autoinduction media and stress media containing ethanol. Negligible levels of protein were observed for all media.
From our efforts to express fluorescent fusion constructs of CRY2 and CIB1 for the aim of conducting a FRET assay, it was discovered that a CRY2 fusion protein has poor expressability in E. coli. Despite the successful expression of the CIB1-YFP construct, a lack of blue fluorescence in the CFP-CRY2 construct suggests the protein may have misfolded or have been proteolysed by the E. coli.