Green Fluorescent Proteins: Towards Extra-Cellular Applications?

Abstract

The Green Fluorescent Protein (GFP) and its numerous variants are applied extensively in a multitude of in vivo applications and have been studied in this context at length. In contrast however, the study of GFP’s within the emerging fields of nanoand micro-technology, which offer broader extracellular applications for GFP and its derivatives, has only recently begun to gather momentum. This thesis presents the directed design of a novel series of Enhanced Green and Enhanced Yellow Fluorescent Proteins (EGFP and EYFP respectively), for implementation in extracellular applications such as biosensing and fundamental research into fluorescence protein behaviour. Each parent fluorescent protein (EGFP or EYFP) was altered to display a single solvent exposed reactive sulfhydryl group with varying degrees of connectivity to the internal GFP chromophore. These sulfhydryl groups were introduced into the protein primary structure via point mutation to yield cysteine residues in place of the targeted native amino acid. Careful examination of the EGFP and EYFP tertiary structures to identify amino acids within the protein primary sequence that fulfilled specific criteria, which were defined in our experimental design, resulted in substitution of amino acids at positions 221, 223, 219, 212 and 97 in EGFP and 221, 223, 212, 95 and 21 in EYFP. Critical development of supporting methodologies delivered vast improvements on literature protocols for expression and purification of the GFP variants listed above. Expression protocol investigation determined that the most prolific E. coli strain for recombinant fluorescent protein production was BL21, which, coupled with our methodology, produced up to 13.6 mg of fluorescent protein per gram of wet cell pellet. The novel purification procedure described in this Thesis delivered highly pure protein with impressive yields (75-80 %). Characterisation of the novel proteins that were designed and produced during this work revealed no change in the proteins’ ability to resist denaturation resulting from cysteine substitution. Neither was there any change in fluorescence emission or UV absorption profiles for standard concentrations (< 60 mM) of any of the purified proteins that were produced. While standard protein solutions returned normal fluorescence emission profiles, solutions that contained protein concentrations above 60 mM displayed red shifted emission maximum values. For protein solutions within the mM concentration range this red shift in fluorescence emission was at times in the order of 30 nm resulting in emission maximums of up to 540 nm for EGFP, and 548 nm for EYFP and recombinant proteins containing an L221C mutation. Preliminary investigations into this phenomenon showed that the changes observed in fluorescence emission were dependent on protein concentration and could be due to dipole-dipole interactions which may be induced by protein aggregate formation at high protein concentrations. Manipulations that were performed on fluorescent proteins during this study included proteolytic digestion with Proteinase K and subsequent testing of the digested protein product. This work identified an increase in the quantum yield of proteolytically digested EGFP and EYFP from 0.6 to 0.8 accompanied by no reduction in the digested proteins resistance to denaturing treatments except when treated with 1 % SDS solution.