Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase

Abstract

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.

Seamless GFP and GFP-Amylase Cloning in Gateway Shuttle Vector, Expression of the Recombinant Proteins inE. ColiandBacillus Megateriumand Assessment of the GFP-Amylase Thermostability

“…This was most probably due to the decrease in the fluorescence quenching from the cellular proteins present in the sample. the obtained results differ from those reported by Zhang et al (29) that the fluorescence intensities of both GFP and GFPthermostable xylanase are thermally sensitive. the authors studied partial length genes.…”

“…A possible explanation for the discrepancy between their results and ours could be the different GFP variants used in the two studies, GFPmut1 in the study of Zhang et al (29) and GFPuv in our study. this suggestion is also supported by the high GFPuv thermostability observed in the study of Moreau et al (18), where the reported temperature for 50 % aggregation of the GFPuv proteins is estimated to be about 79.6 °c,.…”

“…The fusion of studied proteins with fluorescent proteins is increasingly applied for direct assessment of their heterologous expression and conformation stability under various conditions, including thermal stability (18,29,30). this approach offers an attractive option for comparative characterization of groups of newly cloned genes at a larger scale, including comparison of the expression levels of the corresponding recombinant proteins expressed in common microbial hosts.…”

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

“…T=20-40°C, pH=6-8) have recently been proposed, in domains like cell biology and biotechnology. Namely, in GFP-fused proteins folding [19] and enzyme activity [20] assays, and as a biological indicator in thermal processes [21]. In face of these previous observations, it's clear that a thorough and systematic study of the photophysical properties of GFP is of the utmost relevance, and needs to address distinct medium conditions (e.g.…”

The photophysics of LOV-based fluorescent proteins — new tools for cell biology

“…Therefore, LOVbased FPs could successfully be established within research fields where the size of the fluorescent label is critical. For example, the use of iLOV as alternative fluorescent tag enabled advanced studies of the localization, movement and dynamics of target proteins and viruses 9,10,31,32 . Furthermore, in vivo and in vitro studies demonstrated that, in contrast to GFP-like proteins, LOV-based reporter proteins rapidly gain their fluorescence-active conformation because of their fast folding kinetics and the spontaneous incorporation of the chromophore 14,33 , thereby enabling their application as real-time reporters.…”