A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.

Govindan Raghunathan,Hyun-Jong Paik,Ganapathiraman Munussami,Yong-Sung Kim,Hyojin Moon,Seong Soo A An,Sebyung Kang,Sun-Gu Lee

doi:10.1186/1475-2859-13-68

Abstract

BackgroundInclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology.ResultsIn this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn’t affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100–200 nm by reducing the expression time of the protein in vivo.ConclusionsTo our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

Highlights

Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications
The image analysis with fluorescence intensity color palette showed the distinct difference in distribution of proteins between the Green fluorescent protein (GFP)-hs1 and DL4. These results clearly indicated that the aggregation of DL4 occurred in bacterial cell after the protein translation and folding, confirming that the DL4 was exclusively expressed as intracellular active IBs in E. coli
The functional analysis of the DL4 IBs from 5 hours and 30 min expression by confocal microscopy showed green fluorescence emission and exhibited distinct differences in their particle size diameter (Figure 8C). These results suggested that the size of bio-functional fluorescent DL4 IBs could be reduced through simple modulation of expression times, which might enable the preparation of fluorescent protein nanoparticles

Summary

Introduction

Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. Other examples include the functional IBs for diagnosis, tissue engineering and for nano-medicines [10,11,12]. These examples indicate the importance and potentials of active IBs for research and industrial applications

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