Team:IISc-Bangalore/Characterisation

ECFP Characterisation

Introduction

The Green Fluorescent Protein (GFP), naturally produced by the chemiluminescent jellyfish Aequorea victoria, has emerged as one of the most widely used and exploited proteins in molecular and cell biology.
Since the discovery of this fluorophore, several variants have surfaced which aim at modifying the protein to amplify certain characteristics that suit specific biochemical requirements.

ECFP or Enhanced Cyan Fluorescent Protein (belonging to the family of Cyan Fluorescent Proteins – where the Tyr-66 is replaced by a Trp), a monomeric protein, is an artificial derivative of the naturally occurring green fluorescent protein encoded by the GFP gene of A. victoria.
Generation of mutants of GFP has led to a host of fluorophores with increased stability at experimental temperatures as well as brighter and more sustained fluorescence with reduced maturation time. Fluorescent proteins and their allelic mutants function as ubiquitous and indispensable tools for engineering chimeric proteins, where they act as fluorescent tags. These proteins have been expressed in most known cell types as non-invasive markers. They enable a wide range of applications where they help to trace cell lineages, act as reporters for gene expression and help to study protein-protein interactions.

ECFP is produced by the specific mutagenesis of the GFP gene - resulting in the mutations F64L, S65T, Y66W, N146I, M153T and V163A. ECFP imparts a greenish-blue colour to the cells expressing the protein. Although the fluorescence is not as pronounced as in GFP, ECFP has immense applications in energy transfer processes like FRET (Forster or Fluorescent Resonance Energy Transfer). ECFP is commonly used with EYFP (Enhanced Yellow Fluorescent Protein) as an excellent pair in FRET biosensors for visualizing signalling molecules in a cell with high spatio-temporal resolution. ECFP is quite photostable (t½ = 85.0 s) with a sufficiently high lifespan (3.0 ns) and relatively low maturation time, qualifying it as a suitable fluorophore for various biological experiments. It can also function as a molecular pH sensor for fluorescence lifetime imaging.

Owing to the multiple benefits of ECFP, we selected it as the reporter protein to test the gene expression in our CcaS/R system (BBa_K3165043). The ECFP coding sequence is available in the registry under BBa_E0020 and as BBa_E0022 (BBa_E0020 modified to have an LVA tail for rapid degradation).

We chose to characterise the coding sequence of ECFP (BBa_E0020) using the composite part BBa_I13600 already present in the 2019 Distribution Kit. The composite part BBa_I13600 consists of the following components :



Fig (1) : Gene sequence for ECFP generating BioBrick

For our characterisation part, we focused only on the fluorescent protein ECFP and not on the entire biobrick BBa_I13600.
To characterise ECFP, we conducted the excitation and emission spectra of the fluorophore and did the ammonium sulphate precipitation test to determine the solubility concentration of the protein.

Spectral Characterisation

In order to find the excitation and emission wavelengths of the fluorescent protein, we performed wavelength scans over the fluorescence range of the protein. The excitation and emission spectra of the protein so obtained is given below :



Fig (2) : Excitation and Emission Spectra of ECFP

The excitation λ of ECFP was found out to be 429 nm while the emission λ turned out to be 478 nm

Ammonium Sulphate Protein Purification

Ammonium Sulphate Purification of proteins is a method to isolate the protein of interest, based on the salt concentration at which the protein precipitates. The 'salting-out' of proteins is largely based on the amino acid composition of the protein and the nature and complexity of protein folding. In this assay, we perform several rounds of concentrating the protein lysate with a salt (here, ammonium sulphate) while centrifuging after each round of salt addition. Different proteins precipitate out at different salt concentrations providing an estimate of the solute concentration at which they can be extracted.


Fig (3) : SDS PAGE for ECFP induced and uninduced samples
On running the induced and uninduced ECFP crude protein lysates, on a polyacrylamide gel, we obtain bands between 25 kDa and 32 kDa. ECFP has a molecular weight of around 27 kDa. The SDS PAGE exhibits a band at the required size indicating the presence of the gene of interest. The induced sample has a much more pronounced band in comparison to the uninduced sample, signifying minimal leaky transcription.
On running the ammonium sluphate precipitate fractions on a SDS PAGE, we observe the following :


Fig (4) : SDS PAGE for varying percentage saturation fractions of Ammonium Sulphate

From the SDS gel, we can infer that the ECFP protein precipitates at around 55% - 60% saturation of Ammonium Sulphate. The bands of appropriate sizes can be observed in the lanes containing 55% and 60% saturated ammonium sulphate protein precipitates. Thus, we can get an idea of isolating our protein of interest at a specific concentration of salt by differential precipitation.

References

  1. Tsien, R. Y. (1998). The Green Fluorescent Protein. Annual Review of Biochemistry, 67(1), 509–544. doi:10.1146/annurev.biochem.67.1.509 
  2. Review A Guide to Fluorescent Protein FRET Pairs Bryce T. Bajar, Emily S. Wang, Shu Zhang, Michael Z. Lin and Jun Chu