Superactive β-galactosidase inclusion bodies

https://doi.org/10.1016/j.colsurfb.2018.10.049Get rights and content

Highlights

  • Distinct kinetic features of IBβ-Gal at in vitro conditions were described.

  • IBβ-Gal was more efficient than Sβ-Gal to catalyse lactose hydrolysis.

  • Protein-protein interactions in IBβ-Gal preserved β-Gal activity at extremes pH and T.

  • The structure-function relationship of Dβ-Gal was similar to that of Sβ-Gal.

Abstract

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant β-galactosidase inclusion bodies (IBβ-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IBβ-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis–Menten constant, measured in the IBβ-Gal suspensions. Simultaneously, IBβ-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IBβ-Gal represents a reservoir and packed source of highly active and stable enzyme.

Introduction

Inclusion bodies (IBs) are insoluble protein clusters formed in microorganisms during recombinant protein production in the context of conformational stress [[1], [2], [3]]. These self-assembled structures contain an important proportion of active proteins. The quality and the extent of the aggregate is determined, at least partially, by a combination of experimental variables, like bacterial growth temperature, induction temperature and inductor concentration [3,4] and nature of proteins [1]. It has been reported that inside the IB, a network of partially folded proteins with an amyloid-like structure is formed [5]. Properly folded protein molecules are trapped within this network. By means of infrared spectroscopy it has been demonstrated that more than 6% of the whole population of green fluorescence protein (GFP) in IBs is in the soluble folded form. Furthermore, the functionality of the resolubilized GFP from IBs has also been shown [4].

On the other hand, the recovery of soluble bioactive proteins from IBs has been typically accomplished through the treatment with chaotropic agents or acidic treatments, followed by dilution or dialysis into optimized refolding buffers. In most cases, the process involves the protein unfolding. Commonly, after chemical denaturation recombinant oligomeric proteins do not easily adopt an active conformation [6]. Mild conditions have been applied to solubilized protein molecules, contained in IBs, preserving their native-like structure [4,7,8]

IBs are also considered nanosized protein delivery systems for cell therapy. Due to the observed binding of IBs to mammalian cell membranes it is suggested that therapeutic polypeptides might derive from a slow release of active protein from membrane-anchored IB particles. in vitro studies demonstrate a high porosity of IBs that generates enormous solvent-protein interfaces [9] and also spontaneous release of functional IB protein [10]

Recombinant β-d-galactosidase [EC 3.2.1.23] (β-Gal) with or without a fused protein was produced as IBs [[11], [12], [13]]. It was demonstrated the functionality of the enzyme, and not only the conditions to obtain and isolate IBs but also their structural properties [11,14,15].

β-Gal is a soluble enzyme capable of catalyzing lactose hydrolysis in its constitutive monosaccharides: glucose and galactose [16]. In our laboratory we studied the structure-activity relationship of β-Gal in different conditions. In heterogeneous media like those containing liposomes, and thus lipid-water interfaces, we proved that β-Gal interact with membranes [17,18]. Moreover within the membrane milieu the enzyme displayed a more active configuration associated to the protein trapped in a molten globe-like structure [19]. In macromolecular crowded media β-Gal also exhibited a relaxed conformation and higher thermal stability. In the last case we measured lower catalytic efficiency possibly not associated with the protein structure but with diffusional restrictions [20].

The aim of the present work was to investigate the structure-activity relationship of β-Gal present in IBs as well as the functional and structural stability of these protein aggregates (IBβ-Gal). So, we performed enzyme kinetics and protein conformational analysis. Also structural and stability profiles of IBβ-Gal at different dissolution stages were described. These findings contributed to reveal general patterns for the catalysis modulation and provided useful information to define optimal conditions for future technological developments and applications of IBβ-Gal.

Section snippets

Strains and plasmids

E. coli K-12 W3110, E. coli strain BL 21λ Codon plus, pET-26(+) (Novagen, Madison, Wisconsin, United States). Bacteria strains and plasmid were gently provided by Professor Carlos Argaraña from CIQUIBIC CONICET.

Cloning β-Gal gene

The β-Gal gene was amplified from E. coli K-12 W3110 genomic DNA by PCR using primers β-Gal S1N (5′-CATATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCG-3′) and β-Gal A1 (5′- GGCTCGAGTTTTTGACACCAGACCAACTGGTA-3′) the PCR products were digested with NdeI and Xho I (Promega) and inserted into

IB β-Gal kinetic behavior

When the induction of the protein expression occurs at 37 °C the novel proteins tend to arrange in inclusion bodies [4]. It has been reported the presence of protein molecules with a variety of structural qualities (from native to non-native structures) within the IBs [26,27]. Hence, IBβ-Gal enzymatic activity measured in this context reflects the average behavior of an heterogeneous protein sample. In agreement with previous reports, the IBs sample exhibited enzymatic activity. With the aim to

Conclusions

In this work, we showed an increase in the catalytic efficiency of β-Gal against lactose hydrolysis in the context of an in vitro heterogeneous environment like that of IBβ-Gal. Furthermore, in IBs, β-Gal preserved the activity after preincubation at extreme pHs and temperatures suggesting a favourable contribution of protein-protein interactions. We also demonstrated that, in this supramolecular organisation, the catalytic activity was preserved during a long period of time (until two months

Acknowledgments

This work was partially supported by grants from SeCyT-UNC, ANPCyT and CONICET from Argentina. VN, MAP and JMS are Career members of CONICET.

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