Inclusion Body Aggregates Research Articles

Detergent headgroups control TolC folding in vitro

TolC is the trimeric outer membrane component of the efflux pump system in Escherichia coli that is responsible for antibiotic efflux from bacterial cells. Overexpression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which active proteins are recovered. Here, we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native, oligomeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.

Solubilization and Refolding of Inclusion Body Proteins.

Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major hurdle in the recovery of bioactive proteins from E. coli. Usage of strong denaturing buffers for solubilization of bacterial IBs results in poor recovery of bioactive protein. Structure-function understanding of IBs in the last two decades have led to the development of several mild solubilization buffers, which improve the recovery of bioactive from IBs. Recently, combinatorial mild solubilization methods have paved the way for solubilization of wide range of inclusion bodies with appreciable refolding yield. Here, we describe a simple protocol for solubilization and refolding of an inclusion body protein with appreciable recovery.

Solubilization and refolding of variety of inclusion body proteins using a novel formulation

Formation of protein aggregates as inclusion bodies (IBs) still poses a major hurdle in the recovery of bioactive proteins from E. coli. Despite the development of many mild solubilization buffers in last two decades, high-throughput recovery of functional protein from wide range of IBs is still a challenge at an academic and industrial scale. Herein, a novel formulation for improved recovery of bioactive protein from variety of bacterial IBs is developed. This novel formulation is comprised of 20% trifluoroethanol, 20% n-propanol and 2 M urea at pH 12.5 which disrupts the major dominant forces involved in protein aggregation. An extensive comparative study of novel formulation conducted on different IBs demonstrates its high solubilization and refolding efficiency. The overall yield of bioactive protein from human growth hormone expressed as bacterial IBs is reported to be around 50%. This is attributed to the capability of novel formulation to disrupt the tertiary structure of the protein while protecting the secondary structure of the protein, thereby reducing the formation of soluble aggregates during refolding. Thus, the formulation can eliminate the need of screening and optimizing various solubilization formulation and will improve the efficiency of recovering bioactive protein from variety of IB aggregates.

EXPRESSION OF THE STAPHYLOCOCCUS AUREUS LUKE GENE IN THE ESCHERICHIA COLI BL21(DE3) STRAIN

Two-component leukotoxins are important virulence factors for Staphylococcus aureus. Despite efforts made to study S. aureus leukotoxins, the direct mechanism of action of these toxins during infection has not been determined. However, the observation that deletion of LukED significantly attenuates highly virulent S. aureus strains supports the hypothesis that selective inhibition of LukE / D may be useful in the development of new aspects of S. aureus infection control. For this purpose, this work was carried out to test the expression and obtain a recombinant form of the LukE protein in E.coli cells. The LukE gene was cloned into the pET28-c (+) / GFP vector containing the gfp gene. Two fused genes carrying a hexahistidine tag were expressed in cells of the E. coli BL21(DE3) strain. It was found that the 6His-GFP-LucE protein aggregates in inclusion bodies. 6His-GFP-LucE was washed out of inclusion bodies with high molar urea. The 6His-GFP-LucE protein was purified by metal affinity chromatography. Research results can be applied to obtain recombinant protein including strategies for inhibition of toxin activity.

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine)

BackgroundThe current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated.ResultsExpression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days).ConclusionThe presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

TRIM16 controls assembly and degradation of protein aggregates by modulating the p62‐NRF2 axis and autophagy

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress‐induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62‐NRF2 axis. We show that TRIM16 is an integral part of the p62‐KEAP1‐NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress‐induced aggregate clearance and protects cells against oxidative/proteotoxic stress‐induced toxicity in vitro and in vivo. Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation‐associated diseases such as neurodegeneration.

The Role of Sphingomyelin and Ganglioside GM1 in the Interaction of Polyglutamine Peptides with Lipid Membranes

Huntington's disease is a dominant genetic neurodegenerative disorder associated with motor and cognitive decline, caused by a mutation in the poly-glutamine (polyQ) region near the N-terminus of the huntingtin (htt) protein. Expansion of the polyQ region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The polyQ expansion in htt is flanked by a 17 amino acid N-terminal sequence (Nt17) and a proline-rich (polyP) region. Patients with Huntington's disease have reduced ganglioside GM1 levels and restoring these levels in mouse models improves motor function, suggesting a neuroprotective effect, but with an unknown mechanism. To explore the role of GM1 in the cell membrane environment without downstream metabolic effectors, total brain lipid extract (TBLE) model membranes doped with either ganglioside GM1 or sphingomyelin (SM) were exposed to either a model, synthetic polyQ peptide or a full-length htt-exon1 recombinant protein. The interactions between htt and lipid membranes were measured with a combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy. While addition of GM1 to a TBLE membrane resulted in reduced htt insertion and binding, ganglioside inclusion into vesicles had no measureable effect on htt-induced membrane permeability. Atomic force microscopy studies parallel these findings with similar htt-induced morphological changes albeit with an overall decreased interaction between the protein and the membrane at higher GM1 concentrations suggesting the neuroprotective effect of GM1 may be partially attributed to decreased protein-membrane interactions. Parallel studies performed with sphingomyelin indicate that elevated levels of SM cause increases in htt membrane binding, htt-induced membrane permeability, and morphological and mechanical changes in the bilayer associated with exposure to htt-exon1.

Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli.

Well-established genetic manipulation procedures along with a fast doubling time, the ability to grow in inexpensive media, and easy scaleup make Escherichia coli (E. coli) a preferred recombinant protein expression platform. Human alpha-1 antitrypsin (AAT) and other serpins are easily expressed in E. coli despite their metastability and complicated topology. Serpins can be produced as soluble proteins or aggregates in inclusion bodies, and both forms can be purified to homogeneity. In this chapter, we describe an ion-exchange chromatography-based protocol that we have developed involving the use of two anion-exchange columns to purify untagged human AAT from E. coli. We also outline methods that can be used to determine the inhibitory activity of both AAT in cell lysates and purified AAT. Our protocol for the purification of bacterially expressed AAT yields pure and active protein at 6-7mg/l culture.

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

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.

Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent.

BackgroundFormation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30 % trifluoroethanol in presence of 3 M urea and its refolding into bioactive form.ResultsHuman growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize inclusion bodies of a number of proteins.ConclusionsTrifluoroethanol was found to be a suitable mild solubilization agent for bacterial inclusion bodies. Fully functional, bioactive human growth hormone was recovered in high yield from inclusion bodies using trifluoroethanol based solubilization buffer. It was also observed that trifluoroethanol has potential to solubilize inclusion bodies of different proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0504-9) contains supplementary material, which is available to authorized users.

Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process.

Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

The Role of Membrane Context in the Interaction of Polyglutamine Peptides with Lipid Membranes

Huntington's disease is a dominant genetic neurodegenerative disorder associated with motor and cognitive decline, caused by a mutation in the poly-glutamine (polyQ) region near the N-terminus of the huntingtin (htt) protein. Expansion of the polyQ region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The polyQ expansion in htt is flanked by a 17 amino acid N-terminal sequence (Nt17) and a proline-rich (polyP) region. To investigate the interaction between htt exon1 and lipid membranes, a combination of Langmuir trough techniques and vesicle permeability assays measuring calcein leakage were used to directly monitor the interaction of a variety of synthetic polyQ peptides with total brain lipid extract (TBLE) model membranes doped with specific membrane components. Our data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction is enhanced by the presence of the polyP domain. The exogenous addition of cholesterol, a primary neuronal plasma membrane component reduced in many Huntington's disease models, to the TBLE monolayer caused a notable condensing effect in the membrane at low surface pressures. This resulted in reduced peptide insertion into lipid monolayers and decreased levels of induced vesicle permeability, though the effect does not scale linearly with cholesterol concentration. Results from parallel studies on htt-membrane interaction with sphingomyelin, a major component of membranes in the nervous system, will also be discussed.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3)

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0–10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca2+ and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

Solubilization and refolding of inclusion body proteins.

High-level expression of recombinant proteins in Escherichia coli often results in accumulation of protein molecules into aggregates known as inclusion bodies (IBs). Isolation of properly folded, bioactive protein from IBs is a cumbersome task and most of the times results in poor recovery. The process of recovering bioactive proteins from IBs consists of solubilization of IB aggregates using denaturants, followed by refolding of the solubilized protein. Here, we describe a simple protocol for screening of buffers for solubilization of IB proteins. Various IB aggregate solubilization methods including organic solvents have been described.

Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein.

A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form.

Soluble expression and partial purification of recombinant human erythropoietin from E. coli

Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.

The Role of Protein and Membrane Context in the Interaction of Polyglutamine Peptides with Lipid Membranes

Huntington's disease is a dominant genetic neurodegenerative disorder associated with motor and cognitive decline, caused by a mutation in the poly-glutamine (polyQ) region near the N-terminus of the huntingtin (htt) protein. Expansion of the polyQ region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The polyQ expansion in htt flanked by a 17 amino acid N-terminal sequence (Nt17) and a proline-rich (polyP) region. To investigate the interaction between htt exon1 and lipid membranes, a combination of Langmuir trough techniques and vesicle permeability assays measuring calcein leakage were used to directly monitor the interaction of a variety of synthetic polyQ peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on total brain lipid extract (TBLE) model membranes. PolyQ peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces while the polyP region enhanced this interaction. Our data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the polyP domain. The addition of cholesterol to TBLE model membranes, to determine its role in htt-membrane interactions, resulted in reduced peptide insertion into lipid monolayers and decreased levels of induced vesicle permeability, though the effect does not scale linearly with cholesterol concentration. Results from parallel studies performed with ganglioside GM1 and sphingomyelin to determine their role will also be presented.

The amino acid sequences in the C-terminal region of glucose-1-phosphate thymidylyltransferases determine their soluble expression in Escherichia coli

Two similar genes, dnmL and rmbA in Streptomyces peucetius, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases were expressed in Escherichia coli under similar conditions. While RmbA was expressed in soluble form, DnmL was found as insoluble aggregates in inclusion bodies. The difference in expression of these similar proteins led to investigate into the amino acid sequences of these proteins by sequence alignment, hydrophobicity scale and homology modeling. These analyses showed that the two proteins are different only in the C-terminal sequences. Deletion of C-terminal sequence of DnmL increased the expression level of truncated DnmL. Substitution of C-terminal sequence of DnmL with RmbA also expressed the recombinant protein in soluble form. Finally, mutation of six amino acids in DnmL rendered the protein expressed in soluble form. These results suggested that the soluble expression of the thymidylyltransferases lies in the C-terminal sequences. In conclusion, these methods of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E.coli.

The Role of Protein Context in Disease-Related Huntingtin Protein/Lipid Interface Interactions

Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of a poly-glutamine (poly-Q) region near the N-terminus of the huntingtin (htt) protein. Expansion of the poly-Q region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The poly-Q region of htt is flanked by a 17 amino acid N-terminal sequence (N17) and a region of proline repeats (P11). As the cell membrane has been proposed to play a role in mediating htt aggregation, Langmuir trough techniques were used to investigate the effects of flanking regions on the surface activity and insertion of htt peptide constructs into a lipid monolayer. Four peptide constructs were tested: N17-Q35-KK, N17-Q35-P11-KK, KK-Q35-KK and KK-Q35-P11-KK, where the additional lysine residues were attached to improve solubility. Surface activity was measured to determine the affinity of each peptide for the air-water interface. The constructs containing the N-terminal sequence had higher surface activities compared to those without. Inclusion of the polyproline region with the N-terminal sequence contributed to the highest surface activity. To determine peptide association with a cell membrane, insertion of each peptide into a monolayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) held at a constant surface pressure was also investigated. Peptides with the flanking N-terminus showed appreciable monolayer insertion. The polyproline region only mediated and increased the degree of insertion when the N-terminus was also present. Rates of insertion of the constructs containing the N-terminus were comparable, while those without this region were markedly slower. Experiments were repeated using monolayers of total brain extract to better model a physiological environment with similar results. The amino acid environment surrounding the poly-Q region dramatically affects peptide association with the cell membrane and this may potentially mediate the aggregation process.

Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form

Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100mM Tris buffer containing 6M n-propanol and 2M urea. Around 4mg/ml of r-hGH from inclusion bodies were solubilized in 6M n-propanol-based buffer containing 2M urea. Existence of native-like secondary structure of r-hGH in 6M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6M n-propanol and 2M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.