Native-like Secondary Structure Research Articles

Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in β-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

The role of hydrophobic interactions in the molten globule state of globular protein modulated by surfactants.

In order to highlight the role of hydrophobic interactions in the molten globule (MG) state of globular protein modulated by surfactants, the interactions of bovine α-lactalbumin (α-LA) with alkyl trimethylammonium bromides (CnTAB, n=10, 12, 14, and 16) have been studied by experimental and theoretical techniques. Isothermal titration calorimetry (ITC) showed that the enthalpy changes (ΔH) and area of the enthalpogram increased with increasing the chain length of CnTAB. The result of fluorescence, circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectrum suggested that C10TAB and C12TAB unfolded α-LA partially, C14TAB reconstructed protein with a native-like secondary structure content, and C16TAB induced an MG state α-LA. The SAXS results confirmed that the tertiary structure of α-LA was disrupted by C16TAB forming an MG state complex with a micelle-like structure even at the surfactants concentrations below CMC. As indicated by MD results, the β-domain and unstructured region(s) were involved in the MG state α-LA modulated by CnTAB. This work not only provides molecular insights into the role of hydrophobic interactions in the MG state of a globular protein but also helps understand the mechanism of preparing α-LA based biomacromolecule modulated by hydrophobic interactions.

Folding of the nascent polypeptide chain of a histidine phosphocarrier protein in vitro

The phosphotransferase system (PTS), a metabolic pathway formed by five proteins, modulates the use of sugars in bacteria. The second protein in the chain is the histidine phosphocarrier, HPr, with the binding site at His15. The HPr kinase/phosphorylase (HPrK/P), involved in the bacterial use of carbon sources, phosphorylates HPr at Ser46, and it binds at its binding site. The regulator of sigma D protein (Rsd) also binds to HPr at His15. We have designed fragments of HPr, growing from its N-terminus and containing the His15. In this work, we obtained three fragments, HPr38, HPr58 and HPr70, comprising the first thirty-eight, fifty-eight and seventy residues of HPr, respectively. All fragments were mainly disordered, with evidence of a weak native-like, helical population around the binding site, as shown by fluorescence, far-ultraviolet circular dichroism, size exclusion chromatography and nuclear magnetic resonance. Although HPr38, HPr58 and HPr70 were disordered, they could bind to: (i) the N-terminal domain of first protein of the PTS, EIN; (ii) Rsd; and, (iii) HPrK/P, as shown by fluorescence and biolayer interferometry (BLI). The association constants for each protein to any of the fragments were in the low micromolar range, within the same range than those measured in the binding of HPr to each protein. Then, although acquisition of stable, native-like secondary and tertiary structures occurred at the last residues of the polypeptide, the ability to bind protein partners happened much earlier in the growing chain. Binding was related to the presence of the native-like structure around His15.

Using High Pressure and Alkaline pH for Refolding.

The expression of recombinant proteins as insoluble inclusion bodies (IB) has the advantage to separate insoluble aggregates from soluble bacterial molecules, thus obtaining proteins with a high degree of purity. Even aggregated, the proteins in IB often present native-like secondary and tertiary structures, which can be maintained as long as solubilization is carried out in non-denaturing condition. High pressure solubilizes IB by weakening hydrophobic interactions, while alkaline pH solubilizes aggregates by electrostatic repulsion. The combination of high pressure and alkaline pH is effective for IB solubilization at a mild, non-denaturing condition, which is useful for subsequent refolding. Here, we describe the expression of recombinant proteins in Escherichia coli using a rich medium to obtain high expression levels, bacterial lysis, and washing of the IB to obtain products of high purity, and, finally, the solubilization and high yield of refolded proteins using high pressure and alkaline pH.

Transformation of high moisture extrusion on pea protein isolate in melting zone during: From the aspects of the rheological property, physicochemical attributes and modification mechanism

Plant-based meat analogs are emerging products mainly produced by extruder. To reveal the “black box” processing mechanism of extrusion, the physicochemical and rheological properties of pea protein isolate (PPI) were evaluated at different extrusion temperatures (90 °C, 100 °C, 110 °C, 120 °C and 130 °C) and moisture contents (65% and 70%) by using rheological simulation and stop-operation. The results showed a gel-like PPI paste, which presented shear thinning gel-like behavior and strong elastic characteristics. The weaken strain overshoot behavior of the PPI paste transformed to a strong strain overshoot with temperature increased under large amplitude oscillation. The PPI molecules transformed from a spherical structure to a tangled fibrous network, the absolute zeta potential value increased, and the total SH decreased. The native-like secondary structures of PPI were lost, and the relatively rigid protein structure was broken with increasing temperature. The tertiary structure of the raw material is gradually lost, and the Trp residue is transferred from the spherical structure interior to the surface of the protein molecule. The protein subunits were not decomposed during the extrusion process, and although the thermal stability of the PPI was reduced, new cross-links of higher bond energy could be formed at higher temperatures. Moreover, a higher extrusion temperature and proper moisture content can result in partial unfolding and realignment of extruded PPI.

An Unusual Effect of Hofmeister Series Salts on the Stability of Toxoplasma gondii Ferredoxin NADP+ Reductase.

The ionic interactions play an important role in the stabilization of the native conformation of proteins. Toxoplasma gondii Ferredoxin NADP+ Reductase (TgFNR) remains stable at pH 4.0. However, such modulation of ionic interactions leads to compaction and non-cooperativity in its folding. To gain insights into the role of ionic interactions in the modulation of structure and thermodynamic stability of TgFNR. Protein preparations, circular dichroism and fluorescence spectroscopy were used to determine salt-induced changes in the structure and stability of TgFNR. The kosmotropic salts (sodium fluoride and sodium sulphate) appear to induce the biphasic response on the structure and stability of TgFNR. At pH about 4.0, the addition of low concentrations of kosmotropic salts significantly perturbs the existing native-like secondary structure of TgFNR, whereas higher quantities of salt reversed the denaturing impact. This is a one-of-a-kind situation we are unaware of in any other protein. The urea-induced unfolding of TgFNR in the presence of a low dose of salt (100 mM) drastically affected the protein's thermodynamic stability at neutral pH. The increased salt concentrations, on the other hand, reversed the destabilizing effect. Our findings imply that electrostatic interactions are exceptionally significant for the TgFNR stability, however, render highly unusual behavior of Hofmeister series salts, indicating a possible crucial role of salt bridges in the stabilization of different conformations of the protein.

Topological crossing in the misfolded Tetrahymena ribozyme resolved by cryo-EM

The Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) invitro, which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the Tetrahymena L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 Å, 3.8 Å, 4.0 Å, and 3.0 Å, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme's guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5'-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.

Cryo-EM reveals an entangled kinetic trap in the folding of a catalytic RNA.

Functional RNAs fold through complex pathways that can contain misfolded “kinetic traps.” A complete model of RNA folding requires understanding the formation of these misfolded states, but they are difficult to characterize because of their transient and potentially conformationally dynamic nature. We used cryo–electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the Tetrahymena thermophila group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.

Human growth hormone inclusion bodies present native-like secondary and tertiary structures which can be preserved by mild solubilization for refolding

BackgroundNative-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding.ResultsIn this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield).ConclusionsWe have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.

Recombinant PilS: Cloning, Expression and Biochemical Characterization of a Pil-Fimbriae Subunit.

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.

The effect of non-specific binding of Pd(II) complexes with N-heteroaromatic hydrazone ligands on the protein structure

Previously, the cytotoxic actions of five Pd(II) complexes with bidentate N-heteroaromatic chelators (complexes 1?5) on a palette of several cancer cell lines were investigated. However, the results of the cytotoxic activity did not correlate with the hydrophobic character of the complexes. To gain further insight into the structure?activity relationship, essential for the design of novel potential drugs, other factors, such as non-specific interactions with cellular proteins, have to be taken into account. To explore the potential non-specific influence of the complexes on protein structures, ovalbumin (OVA) was chosen as a model system to mimic cellular non-specific crowding environments with high protein concentrations. A Fourier-transform infrared spectroscopy study implied that the binding of 3 and 4 led to only moderate alternations in the secondary structures of the protein, without the possibility to penetrate into hydrophobic core of the protein and disruption of protein native fold. Contrary, the effect of complex 5 on OVA secondary structures was concentration- dependent. While the lower concentration of complex 5 had no effect on OVA structure, a doubled concentration of complex 5 led to complete disruption of the content native-like secondary structures. The concentration-dependent effect of complex 5 on the changes in secondary structures and considerable increase in the exposure of OVA hydrophobic surfaces to water may be related to a potential crosslinking that leads to OVA aggregation.

Recombinant expression and molecular characterization of buffalo sperm lysozyme-like protein 1

Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.

Improved thermal stability of phycocyanin under acidic conditions by forming soluble complexes with polysaccharides

Phycocyanin, a natural blue-colored protein–chromophore complex, forms insoluble aggregates under acidic conditions and loses blue hue during heating. Targeting these issues, phycocyanin was complexed with six polysaccharides (κ-/ι-/λ-carrageenans, xanthan gum, high-methoxyl pectin, and guar gum), respectively; the colloidal and color behaviors of the complexes were examined at pH 3.0 before and after heating (60 °C/80 °C, 30 min). A simple HCl titration strategy was developed to inhibit the associative phase separation commonly occurs at acidic conditions during complexation. Soluble complexes were achieved with ι-carrageenan, xanthan gum, and high-methoxyl pectin, demonstrating good colloidal stability without phase separation at pH 3.0 before and after heating. Fluorescence analyses suggest that the electrostatic complexation between phycocyanin and the mentioned three polysaccharides were reinforced by hydrophobic interactions. The ι-carrageenan–phycocyanin complex exhibits the best colloidal and color stabilities according to laser diffraction particle size analysis and colorimetry. Electrophoresis suggests that the highly negatively charged carrageenans, particularly ι-carrageenan, display high affinity and entrapment efficiency to phycocyanin. Circular dichroism further reveals the ability of carrageenans to stretch phycocyanin's tertiary structures while maintaining native-like secondary structures, which inhibits color changes in phycocyanin's blue hue resulted from the dense interchromophore couplings between the α-helical coiled-coils. Overall, this work dissects polysaccharide–phycocyanin complexation under common beverage processing conditions and provides fundamental insights into the conformational modifications on phycocyanin imparted by polysaccharide–phycocyanin interactions. The improved colloidal and color stabilities would facilitate future application of phycocyanin in foods/beverages as substitutes of artificial pigments.

The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability

Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell–cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range.

Perturbation of anionic surfactant induced amyloid fibrillation by chemical chaperone: A biophysical study

The formation of amyloid fibrils causes different degenerative diseases. For example, the polypeptide hormone insulin, comprising 51 amino acids, is directly linked to type II diabetes due to the formation of amyloid-like aggregates. Herein, insulin was used as a model protein to evaluate the solubilizing potential of alpha-cyclodextrin (α-CD) on the sodium dodecylbenzene sulfonate (SDBS) induced amyloid fibril at pH 2.0. Turbidity, Rayleigh light scattering, binding of Thioflavin T dye, and far-ultraviolet circular dichroism analyses were used to characterize the structural conversion induced by α-CD. ThT binding assay showed the formation of amyloid-like aggregates in the insulin in the presence of 0.5 mM of SDBS. The aggregation was solubilized by α-CD in a dose-dependent manner. At low α-CD concentrations (≤5.0 mM), SDBS-induced amyloid fibrils were not inhibited or solubilized. However, amyloid fibrils completely disappeared or solubilized at α-CD >7.0 mM and the native-like secondary structure of insulin was restored. The results clarify the molecular mechanism underlying the effect of α-CD on amyloid fibrillation of insulin induced by SDBS.

Structural and Functional Characterisation of the Domains of Ubiquitin-Activating Enzyme (E1) of Saccharomyces cerevisiae.

Ubiquitin-activating enzyme (E1) is the first enzyme of the ubiquitination pathway and is required to activate ubiquitin. E1 of Saccharomyces cerevisiae is a large multidomain monomeric protein. There are no studies available on the domains of yeast E1 as independent entities. Four domains of E1 namely, first catalytic cysteine half-domain (FCCH), four-helix bundle (4HB), second catalytic cysteine half-domain (SCCH) and ubiquitin fold domain (UFD) were characterised to understand their structural and functional independence vis-a-vis full length E1. Spectroscopic characterisation using circular dichroism and fluorescence suggested that these domains can act as independent folding units and attain native-like secondary structure. The structural features obtained with the peptides SCCH and FCCH of S. cerevisiae bear a high degree of structural similarity to the corresponding fragments of mouse from literature. Nearly 50% of the residues of the 4HB domain of the S. cerevisiae sample showed helical conformation. They displayed a high degree of conservation when compared with 4HB of mouse with respect to their identity and arrangement. The fragment UFD of yeast formed an α/β domain as in the whole protein and exhibited 45% homology with that of mouse, showing a similar arrangement of α and β elements in its secondary structure. Overexpression of the domains in vivo indicated that the SCCH domain and to some extent UFD apparently interfere with cellular functions such as survival under various stresses.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich "β form" in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

FTIR Investigation of Secondary Structure of Reteplase Inclusion Bodies Produced in Escherichia coli in Terms of Urea Concentration.

Recent studies suggest that reducing the induction temperature would improve the quality of some recombinant inclusion bodies (IB) by providing a native-like secondary structure and leading to an improvement in protein recovery. This study focused on optimizing the solubilization condition of Reteplase, a recombinant protein with 9 disulfide bonds. The influence of lowering induction temperature and urea concentration was investigated on the secondary structure of the recombinant protein through FTIR analysis. Induction temperature reduction decreased the percentage of helixes and loops from 49 to 8. In addition, FTIR spectroscopy corroborates the drastic impact of this parameter on Reteplase secondary structure. Even though lowering urea concentration tripled the solubility of IBs expressed at lower induction temperature, the final yield is still quite low to be considered as optimum. On the other hand, the percentage of beta strands and turns in secondary structure of dissolved proteins were proportional to urea concentration. Therefore, in case of Reteplase, protein expression at low temperature (25 °C) was not efficient to improve the protein recovery yield. Future studies need to focus on using other methods of solubilizing IBs to improve protein recovery.

HBc-based virus-like particle assembly from inclusion bodies using 2-methyl-2, 4-pentanediol

• A chimeric Hepatitis B virus core antigen was expressed as inclusion body. • Sodium dodecyl sulfate dissolved protein retained native-like secondary structure. • Virus-like particles assembled in SDS/2-methyl-2, 4-pentanediol system. Hepatitis B virus core antigen (HBc) has recently been used as carriers to develop recombinant vaccines. However, not virus-like particles (VLPs) but inactive inclusion bodies are often formed for the chimeric proteins when expressed in Escherichia coli . A novel method for in vitro assembly of chimeric HBc-MAGE3 II from inclusion bodies to VLPs was established in this study. The method utilized 2-methyl-2, 4-pentanediol (MPD), an amphipathic di-alcohol, to dissociate sodium dodecyl sulfate (SDS) from the solubilized chimeric protein to initiate VLP assembly. The HBc-MAGE3 II could assemble into VLPs only when the molar ratio of SDS/protein subunit was less than 0.14. After removing SDS/MPD by desalting and further purification, VLPs with similar morphology to the natural virus were obtained. This method could be used for preparation of other VLPs expressed as inclusion bodies.

Inclusion Bodies of Recombinant OmpF Porin from Yersinia pseudotuberculosis: Properties and Structural Characterization.

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.