Abstract
Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the ε-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems.
Highlights
In the fields of biochemistry and structural biology, the green fluorescent protein (GFP)-fusion system with the protein of interest is often used to evaluate the conditions that are suitable for sample preparation
In the course of our biochemical study utilizing GFP, we accidentally found that two distinct protein bands of GFPuv were separated on an SDS-polyacrylamide gel electrophoresis (PAGE) gel after the recombinant expression in Escherichia coli under a conventional condition often used in biochemical studies
We found that one of the molecular species showed the conversion of the ε-amino group into a hydroxyl group in Lys 238 residue at the C terminus, which is conserved in many of the GFP variants used in life science research
Summary
In the fields of biochemistry and structural biology, the GFP-fusion system with the protein of interest is often used to evaluate the conditions that are suitable for sample preparation. The SDS-PAGE analysis in which the samples are subjected to without heat denaturation is useful to assess the molecular weight and integrity of the fusion protein by detection of the fluorescent bands[13,14,15]. GFP is utilized for topology mapping of membrane proteins[16] or protein solubility assessment using split GFP17 These methods, based on the fluorescence of GFP, are useful because they allow us to screen the expression constructs, as well as the conditions of the sample preparation for biochemical and structural analyses with high-throughput using a small amount of samples. In the course of our biochemical study utilizing GFP, we accidentally found that two distinct protein bands of GFPuv were separated on an SDS-PAGE gel after the recombinant expression in Escherichia coli under a conventional condition often used in biochemical studies. The results obtained in this study provided noteworthy information on the utilization of GFP, especially for clarifying the biochemical properties of GFP and its fusion proteins
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