Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers

Overexpression of recombinant N-acetyl-d-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08 ± 0.02 U/mg, was similar to that of the native protein, 2.13 ± 0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41 ± 10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32 ± 5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.     

Solubilization and regeneration of Vitreoscilla hemoglobin isolated from protein inclusion bodies

Vitreoscilla hemoglobin (VHb), a homodimeric protein containing two heme groups in its native state, was used as a model to investigate inclusion body approtein solubilization, prosthetic group incorporation, and reactivation. High-level expression in recombinant Escherichia coli results in accumulation of a substantial portion of heme-free VHb in inclusion bodies. VHb can be solubilized from these inclusion bodies by relatively low concentrations of urea with the dissolution midpoint at approximately 3.2M urea. Dissolution in the presence of stoichiometric heme shifts the dissolution midpoint to approximately 4.5M urea without influencing the dissolution properties of contaminant proteins, suggesting the effect is specific for VHb. Denaturation of apoVHb and holoVHb obtained from purified native VHb has midpoints of 2.9M and 5.1M urea, respectively. VHb solubilized from inclusion bodies with urea at concentrations from 0 to 3.5M urea can be regenerated by heme addition without dilution of urea to yield active holoVHb. The fraction of solubilized VHb reconstituted upon heme addition is maximum at around 30% when solubilization and reconstitution is conducted in less than 1M urea. At these low urea concentrations, approximately 5% of inclusion body VHb is solubilized. These results show the utility of prosthetic group addition to reconstitute holoVHb in the presence of urea. Also, these findings suggest that some inclusion body protein has partially folded conformation and that a fractional dissolution and refolding process may be advantageous.     

Expression, production, and characterization of full-length vitronectin in Escherichia coli

Vitronectin (VN) is one of the primary adhesive proteins in serum and serves to promote the attachment and spreading of a wide variety of cell types to tissue culture plastic. In this study, the pGEX2t expression vector was used to express full-length human VN as a GST-tagged fusion protein in Escherichia coli. GST/VN production was induced with IPTG and the protein was found to localize to inclusion bodies. The inclusion bodies were isolated from cell lysates, washed once with 2 M urea and Triton X-100, and then solubilized with 8 M urea in the presence of a reducing compound. Solubilized GST/VN was purified by heparin affinity chromatography and refolded by dialysis against phosphate buffered saline. Approximately 40 mg of GST/VN was recovered from 1L of bacterial culture. Purified GST/VN migrated at the predicted molecular mass on SDS-PAGE and was recognized by both anti-GST and anti-VN antibodies. GST/VN bound to heparin and promoted cell adhesion, spreading, and growth to a similar extent as that observed with plasma-derived VN. As such, the production of recombinant VN in bacteria represents a rapid and convenient method to produce large quantities of VN for cellular studies.     

Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods.     

Improved solubilization of recombinant human growth hormone inclusion body produced in Escherichia coli

Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGH-dependent cell line.     

Thermolabile antifreeze protein produced in Escherichia coli for structural analysis

Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100 mM Tris buffer containing 6 M n-propanol and 2 M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6 M n-propanol-based buffer containing 2 M urea. Existence of native-like secondary structure of r-hGH in 6 M 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 6 M n-propanol and 2 M 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 6 M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.     

HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。     

Analysis of the disulfide bonding pattern between domain sequences of recombinant prochymosin solubilized from inclusion bodies

Prochymosin contains three disulfide bonds linking Cys45 to Cys50, Cys206 to Cys210, and Cys250 to Cys283. To analyze the disulfide bonding pattern between domain sequences in the recombinant prochymosin molecule solubilized from inclusion bodies by 8 M urea (designated as solubilized prochymosin), a simple peptide mapping method was established. This process consists of thiol alkylation, cleavage with cyanogen bromide, diagonal electrophoresis on polyacrylamide gel, and N-terminal sequencing. By using this procedure it was found that Cys45 and Cys50 located in the N-terminal domain are not mispaired with the cysteine residues, located in the C-terminal domain, in the solubilized wild-type prochymosin and its mutants. This result implies that Cys45 and Cys50, the partners of a native disulfide, are restricted in some ordered structures existing in inclusion bodies and remaining after solubilization. These native structural elements act as folding nuclei to initiate and facilitate correct refolding. The strategy of preserving the native-like structures including native disulfide in the solubilized inclusion bodies to enhance renaturation efficiency may be applicable to other recombinant proteins.Both authors contributed equally to this work     

Purification and characterization of recombinantStreptomyces clavuligerus isopenicillin N synthase produced inEscherichia coli

Recombinant isopenicillin N synthase fromStreptomyces clavuligerus was produced in the form of inactive inclusion bodies inEscherichia coli. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml^–1 gave maximal recovery of active isopenicillin N synthase. Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column. Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20°C, and accounted for about 20% of the total soluble protein. This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps. Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis δ-endotoxins

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry 1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectagular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.     

Refolding and characterization of methionine adenosyltransferase from Euglena gracilis

Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 μmol/min/mg and K(m) values of approximately 85 and 260 μM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed β-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.     

Optimized procedures for purification and solubilization of basic fibroblast growth factor inclusion bodies

The efficiency of purification of basic fibroblast growth factor (bFGF) inclusion bodies using EDTA and nonionic detergents was improved from 25 to 40% by shifting the pH from 8.5 to strong alkaline conditions (pH 9.5 – 10.5). Complete dissolution of bFGF inclusion bodies by guanidinium hydrochloride (> 3 m) was independent of pH and the presence of reducing agents. In contrast, solubilization of bFGF inclusion bodies by urea was pH-dependent and increased in efficiency (e.g. from 0 to 100%) by increasing the pH (from pH 5.0 to 10.5 at 9 m urea). The purification and solubilization procedures are efficient for inclusion body concentrations corresponding to 10 and 100 g per l dry cell weight, respectively.     

Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The beta-strand-rich single chain Fv fragment (scFv) and alpha-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is alpha-helix rich, are predominated by alpha-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the alpha-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.     

Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin

Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.     

Non-reducing alkaline solubilization and rapid on-column refolding of recombinant prion protein

Mature prion protein (PrP) is a 208-residue polypeptide that contains a single disulfide bond. We report an alternative method to purify recombinant mouse PrP produced in Escherichia coli. Bacterial inclusion bodies were solubilized in a buffer containing 2 M urea at pH 12.5. The solubilized protein was rapidly purified on a nickel affinity column without a chaotrope gradient, followed by ion-exchange chromatography. The yield and purity of PrP produced by this alternative approach was similar to that obtained using a conventional solubilization and on-column refolding protocol. Recombinant PrP produced using the non-reducing purification protocol is properly folded, as determined by circular dichroism, and a competent substrate for amyloid fibril formation, as determined by Thoflavin-T dye binding assays. In summary, this report describes a rapid method for producing properly folded recombinant PrP without reducing agents or a chaotrope gradient.     

Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.     

Optimized refolding and characterization of active C-terminal ADAMTS-18 fragment from inclusion bodies of Escherichia coli

The human ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin type-1 modules 18) is a new member of the ADAMTS family. The C-terminal ADAMTS-18 fragment is highly effective at promoting platelet thrombus dissolution in murine model of ischemic stroke, showing significant clinical relevance. In this report, the C-terminal ADAMTS-18 fragment with a GST tag (named rADAMTS-351) was overexpressed mainly as inclusion bodies in Escherichia coli BL21 (DE3) pLysS. The insoluble inclusion body was solubilized and reactivated via a refolding procedure. The optimal buffers for refolding rADAMTS-351 was composed of 50 mM Tris-HCl buffer at pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1 mM DTT, 1 mM GSH, and 0.2 mM GSSG. The refolded rADAMTS-351 was dialyzed and further purified by glutathione-agarose beads. The purity of the final product reached 98% as evaluated by SDS-PAGE and Coomassie Brilliant Blue R250 staining. The recombinant protein displayed its immunoreactivity with anti-C-terminal ADAMTS-18 antibodies by Western blotting. Mass spectroscopic analysis indicated a molecular mass of 65,327 Da as theoretically expected. Purified rADAMTS-351 displayed its bioactivity by inducing platelet fragmentation, which ranged from 81-96% compared to active C-terminal ADAMTS-18 standards. The expression and refolding strategy described in this study allows convenient small-scale production of rADAMTS-351 with biological function and therapeutic potential.     

An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan

The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.