Solubilization Of Inclusion Bodies Research Articles

Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in Escherichia coli.

In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space.

Continuous refolding of a biotech therapeutic in a novel Coiled Flow Inverter Reactor

A novel coiled flow inverter (CFI) based plug flow reactor has been developed for continuous refolding of granulocyte colony stimulating factor (GCSF), a biotech therapeutic product. Solubilized inclusion bodies containing the denatured and reduced forms of GCSF were continuously diluted with the refolding buffer using an inline mixing unit. This was followed by protein refolding into a CFI based tubular reactor in which a helical coil was bent at equidistant right angles to cause flow inversion at each bend. This configuration effectively provided substantial cross sectional mixing while maintaining a favourable distribution of residence time. Design of experiments (DOE) based studies was performed to optimize the refolding protocol with respect to redox conditions, pH and dilution ratio. The performance of the continuous refolding protocol has been compared with an optimized batch refolding protocol. It has been demonstrated that enhanced mixing in CFI allows for operation at higher protein concentrations (0.38mg/ml as compared to 0.19mg/ml in batch) and results in comparable purity (84% vs. 83% in batch), thereby resulting in a nearly 15 times increase in productivity. This will result in a significant reduction of costs related to downstream purification as well as no need for the large tank that is otherwise required for dilution based batch refolding. The proposed configuration is likely to perform favourably in other biotech unit operations that require mixing and/or sharp residence time distribution such as precipitation.

Design and Optimization of a Purification Process for MY32/Ls Protein Solubilizing Inclusion Bodies for a New Vaccine Against Sea Lice

Sea lice are the most problematic marine pathogens the salmon industry has to deal with, significantly affecting Europe and America. The worst offenders are genera: Pseudocaligus, Caligus, and Lepeophtheirus. Over €305 million in losses are estimated. Recent results have suggested that subolesin/akirin/myosin32 are good candidate antigens for the control of arthropod infestations such as sea lice. The aim of this study was to design and optimize the purification step of MY32/Ls protein to obtain the active pharmaceutical ingredient against sea lice. Non-chromatographic purification strategies were employed, based on published works, to establish rupture, washing, solubilization, and refolding conditions. A disruption process using a bead mill was established. Cell culture volume was flushed through the mill eight times. Three 60-minute process steps for washing inclusion bodies were performed, and the fourth washing step was a 20-minute process. The first two washes with a Tris-HCl (50 mM) buffer were performed using a non-ionic surfactant (Triton X-100). For the third wash, the surfactant was omitted. The fourth washing step was carried out at a pH of 10.5. The inclusion body solubilization process, in an alkaline condition of pH 12.5, lasted 90 minutes. For the refolding step, Tween 80 was used as a stabilizing additive. The API obtained had a better immune response (IgG) than the positive control, with higher purity.

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.

A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form.

BackgroundMild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies.ResultsHere we presented an efficient method for mild solubilization of inclusion bodies by using a freeze-thawing process in the presence of low concentration of urea. We used two different proteins to demonstrate the advantage of this method over the traditional urea-denatured method: enhanced green fluorescent protein (EGFP) and the catalytic domain of human macrophage metalloelastase (MMP-12_CAT). Firstly, PBS buffer at pH 8 containing different molar concentration of urea (0-8 M) were used to solubilize EGFP and MMP-12-CAT inclusion bodies and the solubility achieved in 2 M urea in PBS buffer by freeze-thawing method was comparable to that of PBS buffer containing 8 M urea by traditional urea-denatured method. Secondly, different solvents were used to solubilize EGFP and MMP-12_CAT from inclusion bodies and the results indicated that a wide range of buffers containing 2 M urea could efficiently solubilize EGFP and MMP-12_CAT inclusion bodies by freeze-thawing method. Thirdly, the effect of pH and freezing temperature on the solubility of EGFP and MMP-12_CAT inclusion bodies were studied, revealing that solubilization of inclusion bodies by freeze-thawing method is pH dependent and the optimal freezing temperature indicated here is −20°C. Forth, the solubilized EGFP and MMP-12_CAT from inclusion bodies were refolded by rapid dilution and dialysis, respectively. The results showed that the refolded efficiency is much higher (more than twice) from freeze-thawing method than the traditional urea-denatured method. The freeze-thawing method containing 2 M urea also effectively solubilized a number of proteins as inclusion bodies in E.coli.ConclusionsMild solubilization of inclusion body proteins using the freeze-thawing method is simple, highly efficient and generally applicable. The method can be utilized to prepare large quantities of bioactive soluble proteins from inclusion bodies for basic research and industrial purpose.

Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris

The large scale screening of cytokine mutants is a component of binding and activity mapping and requires an efficient method of cytokine protein expression. Here, we compared recombinant IL-7 expression with and purification from Escherichia coli and Pichia pastoris. The IL-7 cytokine contains three disulfide bonds that are essential for its biological activity, and which are formed upon secretion through P. pastoris, but not in the reducing cytoplasm of E. coli. In contrast to a previous report we found that P. pastoris secretes active but N-linked hyperglycosylated IL-7. Enzymatic deglycosylation was incompatible with activity measurements in a cell based assay. E. coli expressed IL-7 was refolded from solubilized inclusion bodies. A chromatographic purification step between inclusion body solubilization and refolding increased the yield of biologically active monomeric IL-7, and decreased the amount of inactive soluble aggregates. Cation exchange chromatography of untagged IL-7, and IMAC of His-tagged IL-7 improved refolding yields to a similar extend, indicating that the removal of contaminating components in the solubilized inclusion bodies improves refolding efficiency. We conclude that a chromatographic purification step of IL-7 solubilized from E. coli inclusion bodies increases refolding yield, and may be a suitable general rescue strategy for obtaining folded and biologically active proteins from inclusion bodies.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC–MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.

Identification and immunogenic potential of B cell epitopes of outer membrane protein OmpF of Aeromonas hydrophila in translational fusion with a carrier protein.

Aeromonas hydrophila, a ubiquitous and virulent bacterial pathogen, affects a variety of fishes, including Labeo rohita. Existing treatment strategies comprise antibiotic therapies and attenuated bacterial strain-based vaccines. No functional subunit vaccine has been available until now. Given their key role in determining pathogenicity, outer membrane proteins have been successfully explored as potential vaccine candidates. We have devised a direct strategy for eliminating non-specific responses by selectively aiming the immune response against specific immunodominant epitopes of the outer membrane protein F (OmpF) of A. hydrophila (AhOmpF). Five putative epitopes of AhOmpF predicted in silico were genetically conjugated with heat labile enterotoxin chain B of E. coli (LTB). Recombinant fusion proteins expressed in E. coli were purified from solubilized inclusion bodies and refolded. The fusion protein retained GM1 ganglioside receptor binding activity of LTB, indicating proper folding. Four of the five fusion proteins were found to be highly immunogenic. Of the four proteins, antisera against the fusion protein (anti-rEpiF1) harboring 66-80 amino acid residues of the OmpF gave maximum cross-reactivity with the targeted rOmpF in enzyme-linked immunosorbent assay (ELISA) and was able to recognize both fusion partners-rOmpF and rLTB-in Western blot. Antibody isotyping of the antisera and cytokine array analysis of the culture supernatants of splenocytes from sensitized mice manifested a mixed Th1/Th2 immune response with a bias toward Th2. Anti-rEpiF1 antibodies were able to bind to the cell membrane of live A. hydrophila cells and agglutinate them. Our results thus suggest that the OmpF epitope (66-80) in fusion with a carrier protein is a promising vaccine candidate against A. hydrophila.

Optimization of conditions for high-level expression and purification of human recombinant consensus interferon (rh-cIFN) and its characterization.

Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5kDa and specific activity was 2.0 × 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.

Optimizing Primary Recovery and Refolding of Human Interferon-b from Escherichia coli Inclusion Bodies

Background: The refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. Objectives: The purpose of this study was optimization of recombinant human interferon-b purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity. Materials and Methods: Triton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-b inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, b-mercaptoethanol and dithiothritol. Results: The best recovery was obtained in the presence of 0.5% TritonX-100 (v/v). Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon-b. Successful refolding was achieved by gradient elution (decreasing the guanidine-hydrochloride concentration) in the presence of L-arginine. Partial purification was also achieved continuously, and recombinant human interferon-b was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon. Conclusions: In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg. Conclusion: In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH = 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg.

Effect of post-solubilization conditions on the yield and efficiency of recombinant streptokinase purification at large-scale

Streptokinase, a plasminogen activator which converts plasminogen to plasmin and consequently promotes fibrinolysis, is the leading drug for treating acute myocardial infarction in developing countries and its production is industrially demanded. In this work, the substantial influence of inclusion body (IB) post-solubilization condition on the performance of a sequential chromatography method for large-scale purification of recombinant streptokinase was demonstrated. In the preliminary experiments, various post-solubilization pH conditions were studied, and it was shown that the pH value of solubilized inclusion bodies (i.e., in 4M urea) had a marked impact on the purity of streptokinase obtained at the end of post-solubilization process. When the pH value of the solution containing solubilized IBs was decreased from 7.5 to 6.5 and 6.0, the greatest increases (10% and 27%, respectively) in streptokinase purity occurred. The influence of different post-solubilization pH conditions on the efficiency and yield of large-scale chromatographic purification methods was next investigated. When the solubilized IBs solution with pH adjusted to 6.0 was utilized for subsequent sequential chromatography process, the complete elution peak with high overall yield (91.3%) and purity (98%) was achieved. In comparison to this, while the sequential chromatography procedure was instigated by using the solubilized IBs solution with pH 4.2, four elution fractions (EF1 to EF4) with disparate target protein purities (i.e., 57%, 77.3%, 91.4% and 86.7%, respectively) were attained, the process was incompletely effective, and the highest recovery and purity figures (81.8% and 91.4%, respectively, belonging to EF3) were much lower than those for the earlier process.

Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

Purification, Characterization and Plasma Half-Life of PEGylated Soluble Recombinant Non-HA-Binding CD44

The aim of this study was to increase the serum half-life of recombinant CD44 hyaluronan (HA) binding domain by PEGylation. We have previously found that recombinant soluble CD44 HA binding domain (CD44HABD) and its non-HA-binding triple mutant CD44HABD(R41AY78SY79S) (CD44-3MUT) inhibits angiogenesis and subcutaneous tumor growth. However, this ~12 kDa recombinant protein displays a high serum clearance rate. Here, we report the purification of monomeric CD44-3MUT from urea solubilized inclusion bodies using weak anion exchange chromatography and gel filtration. To increase the serum residence time of CD44-3MUT we PEGylated the resulting protein using 20 kDa methoxy-PEG-propionaldehyde. PEGylation of CD44-3MUT prolonged its in vivo serum half-life about 70-fold from 0.03 to 1.8 hours. Along with extended plasma residence time, PEGylation also increased the systemic exposure. By cell impedance assay we confirmed that PEGylated CD44-3MUT maintained its in vitro function. The results from the impedance assay additionally demonstrate that the CD44-3MUT effect on endothelial cells is mediated by vimentin. In summary, we have developed a purification protocol for large-scale production of CD44-3MUT and generated a PEGylated form of CD44-3MUT. HA binding domain of CD44(CD44HABD) and its modified non-HA binding form (CD44-3MUT) inhibit angiogenesis and tumor growth in vivo without disturbing HA-binding functions. CD44-3MUT has been PEGylated for use as a new type of anti-angiogenic human drug. PEGylation of CD44-3MUT improved pharmacokinetic properties but retains its functional activity.

Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has multiple roles in hematopoietic cells such as the regulation of proliferation and differentiation. Here, we describe fed-batch culture, refolding, and purification of rhG-CSF. The suitability of urea or sarcosine for solubilizing inclusion bodies (IBs) was tested. It was observed that urea is more efficient for solubilizing and refolding IBs than sarcosine is. The purity of rhG-CSF and the removal percentage of the rhG-CSF isoforms during purification were increased by pH 5.5 precipitation. The purity and the yield of purified rhG-CSF were 99% and 0.5 g of protein per liter culture broth, respectively. Our protocols of recombinant protein purification using ion exchange chromatography and semipreparative high performance liquid chromatography of pH-precipitated refolded solution may be informative to the industrial scale production of biopharmaceuticals.

Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing

Protein aggregation is a common challenge in the manufacturing of biological products. It is possible to minimize the extent of aggregation through timely measurement and in-depth characterization of aggregation. In this study, we demonstrated the use of dynamic light scattering (DLS) to monitor inclusion body (IB) solubilization, protein refolding, and aggregation near the production line of a recombinant protein-based vaccine candidate. Our results were in good agreement with those measured by size-exclusion chromatography. DLS was also used to characterize the mechanism of aggregation. As DLS is a quick, nonperturbing technology, it can potentially be used as an at-line process analytical technology to ensure complete IB solubilization and aggregate-free refolding.

Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling

An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from Npro fusion pep6His and Npro fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes – one using urea for IB dissolution, the other one using NaOH for IB dissolution – and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes.

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37°C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37°C are more efficiently dissociated at 2.4kbar than those produced at 47°C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69kbar) refolding step. The association of a negative temperature (−9°C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10°C to 50°C is proportional to the temperature, and a higher yield was obtained at 20°C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37°C), of their dissociation at HHP (−9°C) and of eGFP refolding (20°C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.

Prediction of inclusion body solubilization from shaken to stirred reactors

Inclusion bodies (IBs) were solubilized in a µ-scale system using shaking microtiter plates or a stirred tank reactor in a laboratory setting. Characteristic dimensionless numbers for mixing, the Phase number Ph and Reynolds number Re did not correlate with the kinetics and equilibrium of protein solubilization. The solubilization kinetics was independent of the mixing system, stirring or shaking rate, shaking diameter, and energy input. Good agreement was observed between the solubilization kinetics and yield on the µ-scale and laboratory setting. We show that the IB solubilization process is controlled predominantly by pore diffusion. Thus, for the process it is sufficient to keep the IBs homogeneously suspended, and additional power input will not improve the process. The high-throughput system developed on the µ-scale can predict solubilization in stirred reactors up to a factor of 500 and can therefore be used to determine optimal solubilization conditions on laboratory and industrial scale.

Mechanism and model for solubilization of inclusion bodies

Inclusion bodies are a source of large quantities of protein with high purity over-expressed in Escherichia coli. Efficient solubilization is the initial step in recovering the protein. We visualized solubilization using high-resolution light and transmission electron microscopy over time and the most common solubilization agents, urea and guanidine hydrochloride, as well as sodium hydroxide. Upon solubilization, the inclusion bodies were penetrated by the solubilization agent, shrinking the densely packed cores as the protein diffused out. Despite the complex process, solubilization could be described by a homogeneous layer model with a high order of reaction. The solubilization of protein aggregates, such as inclusion bodies, does not follow the rules of ordinary solubilization processes, but is controlled by pore diffusion. Consequently, power input and mixing frequency is not relevant to the process.

Microwave assisted solubilization of inclusion bodies

AbstractBackgroundProduction of recombinant proteins in bacterial hosts often produces insoluble intracellular particles called inclusion bodies. Recovery of active protein from inclusion bodies generally requires their solubilization in chemical denaturants followed by a refolding strategy. The solubilization is carried out with shaking/stirring and takes several hours.ResultsUsing inclusion bodies of seven diverse kinds of recombinant proteins [mutants of controller of cell division or death protein B (CcdB), human CD4D12, thioredoxin fusion protein (malETrx), mutants of maltose binding protein (MBP), single chain variable fragment (ScFv) b12 and single chain antigen binding fragment (ScFab) b12 (anti-HIV-1)], it is shown that exposure to microwave irradiation (200 W) for 2 min, solubilized these inclusion bodies completely. This was confirmed by data based upon turbidity measurements at 400 nm and dynamic light scattering studies. These solubilized inclusion bodies could be refolded correctly in all the cases by known methods. The refolding was confirmed by fluorescence emission spectra and biological activity studies.ConclusionSolubilization of the inclusion bodies before refolding is a part of protein production processes for several recombinant proteins which are overexpressed in the bacterial host systems. Our results show that microwave assistance can considerably shorten the process time.