GFP PURIFICATION

GFP Purification



GFP Purification

Introduction

Green fluorescent protein (GFP), produced naturally by the bioluminescent jellyfish Aequorea victoria and first synthesized from a cloned gene by Prasher et. al. in 1992, has in the past decade become the most widely-used reporter protein in biological and bioengineering applications. Its primary appeal lies in the fact that although it can be detected non-invasively, continuously, and without the addition of any additional substrate or cofactor (Heim et. al., 1994), it provides a clear visual representation of the localization of any protein within a single cell or multicellular system. Historically, GFP has been used only for imaging applications—using recombinant DNA technology, it is possible to attach it to a target protein, forming a biologically-active “chimeric” complex that retains the functionality of the target but also fluoresces green when exposed to light of wavelength 395 nm (Chudakov, 2005, 605).

Materials and Methods

In each case, the purification technique of Paramban et. al. is only tangentially related to the content of the paper as a whole; no paper has yet used it directly for one of the many conceivable biological or bioprocess applications. Indeed, comparatively little time has passed since the original paper was first published, perhaps explaining the relative scarcity of papers on the subject. However, the practice and study of other techniques involving GFP have, in general, grown so quickly as to suggest some inherent limitation in the purification technique of Paramban et. al. preventing it from achieving more widespread usage and citation in the literature. This paper seeks to explicitly explore possible applications of 6xHis GFP IMAC purifications to biotechnology and speculate on the possibilities of why these applications have not yet been clearly adopted by contemporary researchers.

GFP consists of 11 antiparallel strands of amino acid monomers forming a ?-barrel which contains an aromatic chromophore derived from intramolecular reactions between Serine-65 and adjacent residues (Tsien, 1998). The chromophore itself, a phenol in wild-type GFP (GFP-wt), strongly absorbs ultraviolet light of wavelength 395 nm to reach an excited state from which it emits a distinct green light of wavelength 508 nm. Molecular engineering efforts over the past decade have resulted in synthetic GFP derivatives with drastically improved fluorescence properties.

Results

The fraction of GFP fluorescence under UV light; the results are presented in table 1. Table 1.1, demonstrates a graph which produced distance migrated vs. molecular mass.

To purify the GFP, the solution was transferred to microcentrifuge tubes were spun ...