Characterization on the aggregation of self-aggregating green fluorescent protein variant

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Abstract

s-DL4 is a variant of green fluorescent protein (GFP), exclusively deposited in vivo into active inclusion bodies (IBs). In this study, we demonstrated that s-DL4 is a self-aggregating molecule by performing structural analysis of s-DL4 IBs and studying in vivo/in vitro aggregating properties of the molecule. Fourier transform infrared analysis of IBs revealed that there were native GFP structures and intermolecular interactions between the protein molecules. s-DL4 was always deposited into insoluble intracellular IB aggregates, regardless of the protein expression rate. The active s-DL4 IBs dissolved in urea solution were aggregated and precipitated when the urea was removed by dialysis.

Introduction

Production of heterologous proteins in bacterial cells often results in the formation of inclusion bodies (IBs), which are pseudospherical particles, ranging from nanometers to micrometers in diameter. In general, IBs represent misfolded and inactive protein deposits, which have been considered to be waste by-products of protein expression. However, this viewpoint is changing because IBs are protein particles which have some advantages over conventional synthetic polymer-based particles. IB protein particles composed of natural amino acids and peptide bonds are very biocompatiable, and therefore they are of great interest in biomedical research. For instance, they can be used as novel materials for stimulating mammalian cell proliferation in tissue engineering studies and as vehicles for therapeutic proteins in advanced cell therapy [1], [2], [3], [4]. They can be ecofriendly produced by conventional recombinant DNA technology and fermentation technology. In addition, protein engineering tools allow us to fabricate their physical and chemical properties very simply. Recently, it was reported that even the activity of a protein can be retained in IB particles by protein engineering [5], [6], [7], [8].

Green fluorescent protein belongs to a family of fluorescent proteins that are functionally active when the β-barrel structure, composed of 11 β-stands and a single central helix, is properly folded. A few studies have demonstrated that active GFP IBs could be obtained by fusing GFP and peptide or protein sequences with self-aggregation/assembly properties [9], [10], [11]. It has been proposed that such active GFP IBs can be good fluorescent particles or precursors for the preparation of biomaterials.

We were recently able to obtain a GFP variant exclusively deposited to intracellular active IBs [12], [13]. The mutant s-DL4 devoid of an exposed loop sequence (191-GPVLLP-196) was discovered in the process of studying the effects of loop deletion mutations on GFP folding and activity [12]. The mutant was expressed in insoluble form with fluorescent activity when produced in Escherichia coli, indicating the possibility of preparing fluorescent protein particles on a large scale. Their morphologies and sizes were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) [13]. The study was the first example to demonstrate that active IBs can be generated by engineering an intrinsic protein sequence without tagging any peptide or protein sequences showing self-aggregation/assembly properties.

In our previous studies, it was demonstrated that spectral properties, quantum yield, refolding rate, and specific fluorescent activity of s-DL4 were similar to those of its native form, and s-DL4 IBs were found to be composed of active s-DL4 proteins. Considering these results, it was assumed that s-DL4 was properly folded and that folded s-DL4 molecules might have aggregated in the cell. However, the studies focused on the demonstration of intracellular formation of active IBs, and therefore, the possibility that some artifacts may have caused intracellular aggregation could not be ruled out.

In this study, the hypothesis that s-DL4 has intrinsic self-aggregating properties, which leads to the formation of active intracellular aggregates, was confirmed by characterizing s-DL4 IBs using several approaches. First, the protein structure and intermolecular interactions in s-DL4 IBs were studied by Fourier transform infrared spectroscopy (FTIR). Second, in vivo aggregation of s-DL4 was tested under various expression conditions that are capable of inhibiting protein aggregation. Finally, s-DL4 molecules were dissociated from IBs and reaggregated to confirm their in vitro aggregation properties.

Section snippets

Expression and purification of s-DL4 IBs

Gene variants encoding mutant (s-DL4), cloned in the pET30b vector, were expressed in E. coli BL21 (DE) and purified as previously described [13]. Briefly, IBs of DL4 were purified by collecting and washing the insoluble pellet.

Secondary structure analysis by FTIR

The insoluble pellet was purified by repeated washing using a wash buffer (50 mM Tris, 50 mM NaCl, 1% Triton X-100, 1 M urea, pH 8.0) and dried in a Speed-Vac system for 40 min prior to analysis to reduce water interference in the spectrum. The spectrum was recorded on an

Structural characterization of s-DL4 aggregates by FTIR

The assumption that s-DL4 IBs are formed by intermolecular interactions between properly folded, active s-DL4 molecules was based on our previous observation that s-DL4 is mostly composed of active proteins [12], [13]. To demonstrate the assumption, the protein structure and intermolecular interactions in s-DL4 were investigated by FTIR. FTIR is a powerful tool for studying structural characteristics of aggregated proteins and has been extensively used to characterize protein structures and

Conclusion

s-DL4 is a GFP variant exclusively deposited into intracellular active IBs. In our previous studies, it was assumed that s-DL4 was properly folded and that folded s-DL4 molecules might have aggregated. However, the possibility that some artifacts may have caused intracellular aggregation could not be ruled out. In this study, the hypothesis that s-DL4 has intrinsic self-aggregating properties was confirmed by characterizing s-DL4 IBs. It is expected that the confirmed self-aggregating property

Acknowledgment

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01056766).

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