Abstract
Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.
Highlights
Proteins are often chemically modified by covalent conjugation to investigate the conformational dynamics, molecular interactions and enzyme kinetics in order to gain insight into their biological functions [1,2]
Size-exclusion chromatography is commonly used for the removal of unreacted Fluorescein isothiocyanate (FITC) molecules; this process is accompanied with the loss of proteins and undesired dilution of the sample, especially c 2018 The Author(s)
We have modified the commercially available pMALc5E plasmid vector to enhance protein homogeneity, concentration, and labelling efficiency. We accomplished this by introducing maltose-binding protein (MBP) at the N-terminus and His6 tag at the C-terminus regions of the target protein
Summary
For TAP tag, modify pMALc5E vector by introducing TEV recognition cleavage-site between N-terminus of MBP and MCS. Insert His tag at the C-terminal region of the vector (Figure 1B). Materials required Escherichia coli DH5α (New England Biolabs (NEB), Ipswich, MA, U.S.A.), pMAL c5E (NEB), Plasmid miniprep kit (Sigma chemicals, St. Louis, MO, U.S.A.), Quick-change site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, U.S.A.), Ampicillin sodium salt (Sigma), Primers (Sigma), DpnI enzyme (Fermentas, Waltham, Massachusetts, United States), IPTG (Sigma), Luria-Bertani (LB) broth (Himedia, Mumbai, India)
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