Urea Gradient Research Articles

Urea-Gradient Protein Refolding in Size Exclusion Chromatography

Protein refolding using urea gradient size exclusion chromatography (UGSEC) is a new version of conventional SEC refolding, which incorporates urea gradient into the SEC. Operating factors, mainly the urea gradient length and the final urea concentration in the gradient, are discussed in detail. Recent applications of UGSEC, including refolding of recombinant human granulocyte colony stimulating factor by UGSEC, is shown as an example.

Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.

Heterologous expression, purification and characterization of the influenza A virus M2e gene fused to Mycobacterium tuberculosis HSP70359–610 in prokaryotic system as a fusion protein

One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70(359-610)), we can render it very immunogenic. According to previous reports, this study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e, a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70(359-610), as a carrier and adjuvant. We fused the genes of M2e and HSP70 ( 359-610 ) then inserted in pQE-60, prokaryotic expression vector. This recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M-15. The recombinant fusion protein was purified by Ni-NTA affinity chromatography under denaturing conditions, followed by urea gradient dialysis. The purified fusion protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial penta-His HRP conjugate antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on the infected MDCK cells surface by immunofluorescence and cell-ELISA assay using rabbit's immunized antiserum. This observation suggest that the expressed fusion protein is useful as a universal recombinant vaccine for overcoming highly mutational influenza virus, but more immunological study in animal lab remains to be evaluated.

Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS

The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography (WAX) coupled with MALDI-TOF MS. The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX. However, with the urea gradient elution and in the presence of GSSG/Cyst as the ratio 1:6 in the mobile phase employed, the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the correct three disulfide bonds. The protein refolding efficiency of reduced/denatured insulin can be increased from 3% to 34%. The effects of urea gradient and the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst, on the forming the disulfide bonds of reduced/denatured insulin were investigated in detail. The results were further tested by the separation of the WAX fraction of reduced/denatured insulin with RPLC and MALDI-TOF MS.

Expression of β-agarase I DagA in prokaryotic cell and its activity identification

AbstractThe DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA ofPseudoalteromonas atlantica19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed inEscherichia coliER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.

A diffusion-adjusted regional blood flow model to predict solute kinetics during haemodialysis

Sequestration of creatinine, in both erythrocytes and other cells, has complicated the widespread application of creatinine kinetics in haemodialysis. The goal of this study was to determine whether creatinine kinetics could be described using a regional blood flow (RBF) model that also incorporated diffusion between intra- and extracellular fluids. Transport between intra- and extracellular spaces was modelled by diffusion using a specific rate constant k(s) for creatinine equilibration in whole blood (0.022 min(-1)) determined in a separate study. This k(s) was applied to all body spaces and to creatinine removal from blood coursing through the dialyzer. Erythrocyte and plasma creatinine and urea concentrations during haemodialysis measured and reported by others were used to test the model. The model accurately predicted the reported time course of creatinine in plasma and erythrocytes as well as the time course of urea in plasma when using the much higher k(s) for urea (158 min(-1)). However, it did not explain an increased erythrocyte to plasma urea gradient found at the end of haemodialysis. The results suggest that a diffusion-adjusted regional blood flow (DA-RBF) model can be used to explain compartmentalization of creatinine or urea throughout the body during haemodialysis, although possible additional compartmentalization of urea in erythrocytes, and perhaps in the tissues, still needs to be accounted for. This new model should be applicable to modelling of other non-protein-bound candidate uraemic toxins, also.

Refolding and purification of rhNTA protein by chromatography

RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size-exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size-exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two-gradient size-exclusion chromatography refolding processes, the refolded rhNTA was purified by ion-exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS-PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg.

High Recovery Refolding of rhG-CSF from Escherichia coli, Using Urea Gradient Size Exclusion Chromatography

Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.

Improving the refolding of NTA protein by urea gradient and arginine gradient size-exclusion chromatography

Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme.

Continuous monitoring of the bronchial epithelial lining fluid by microdialysis

BackgroundContents of the epithelial lining fluid (ELF) of the bronchi are of central interest in lung diseases, acute lung injury and pharmacology. The most commonly used technique broncheoalveolar lavage is invasive and may cause lung injury. Microdialysis (MD) is a method for continuous sampling of extracellular molecules in the immediate surroundings of the catheter. Urea is used as an endogenous marker of dilution in samples collected from the ELF. The aim of this study was to evaluate bronchial MD as a continuous monitor of the ELF.MethodsMicrodialysis catheters were introduced into the right main stem bronchus and into the right subclavian artery of five anesthetized and normoventilated pigs. The flowrate was 2 μl/min and the sampling interval was 60 minutes. Lactate and fluorescein-isothiocyanate-dextran 4 kDa (FD-4) infusions were performed to obtain two levels of steady-state concentrations in blood. Accuracy was defined as [bronchial-MD] divided by [arterial-MD] in percent. Data presented as mean ± 95 percent confidence interval.ResultsThe accuracy of bronchial MD was calculated with and without correction by the arteriobronchial urea gradient. The arteriobronchial lactate gradient was 1.2 ± 0.1 and FD-4 gradient was 4.0 ± 1.2. Accuracy of bronchial MD with a continuous lactate infusion was mean 25.5% (range 5.7–59.6%) with a coefficient of variation (CV) of 62.6%. With correction by the arteriobronchial urea gradient accuracy was mean 79.0% (57.3–108.1%) with a CV of 17.0%.ConclusionUrea as a marker of catheter functioning enhances bronchial MD and makes it useful for monitoring substantial changes in the composition of the ELF.

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.

Aquaporin 1, Urea Transporters, and Renal Vascular Bundles

Early theories of urinary concentration focused upon export of sodium by the thick ascending limb of Henle as the “single effect” that, when multiplied along the medullary axis, generates high osmolarity to extract water from the collecting duct. To account for the presence of a urea gradient

Genetic microheterogeneity of human transthyretin detected by IEF

Mutations of the human transthyretin (TTR) gene have attracted medical interest as a cause of amyloidosis. Recently, we have described in detail an electrophoretic procedure with PAGE followed by IEF in urea gradients for the study of the microheterogeneity of TTR monomers (Altland, K., Winter, P., Sauerborn, M. K., Electrophoresis 1999, 20, 1349-1364). In this paper, we present a study on 49 different mutations of TTR including 33 that result in electrically neutral amino acid substitutions. The aims of the investigation were to test the sensitivity of the procedure to detect TTR variants in patients with TTR amyloidosis and their relatives and to identify some common characteristics that could explain the amyloidogenicity of these variants. We found that all tested amyloidogenic mutations could be detected by our method with the exception of those for which the corresponding variant was absent in plasma samples. Most of the electrically neutral amyloidogenic TTR variants had in common a reduced conformational stability of monomers by the activity of protons and urea. For three variants, e.g. TTR-F64L, TTR-I107V and TTR-V122I, the monomers had a conformational stability close to that of normal monomers but we found experimental and structural arguments for a weakening of the monomer-monomer contact. All types of amyloidogenic mutations affected the stability of TTR tetramers.

Detection and quantification of Vibrio populations using denaturant gradient gel electrophoresis

Bacteria affiliated with the genus Vibrio are endemic in marine and estuarine ecosystems and are also found in many freshwater environments. Vibrios can enter viable but non-culturable states and since many species are pathogenic, there is a great need for culture-independent methods that identify and quantify multiple Vibrio populations. We adopted Vibrio-specific 16S rRNA-directed primers and a competitive PCR protocol (QC-PCR; [Thompson, J.R., Randa, M.A., Marcelino, L.A., Tomita-Mitchell, A., Lim, E., Polz, M.F., 2004b. Diversity and dynamics of a North Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70, 4103–4110]) for separation and quantification of Vibrio populations using denaturant gradient gel electrophoresis (DGGE). Sixteen Vibrio isolates and eight environmental samples were used to assess the precision and resolution of the method. A 45–70% gradient of Urea and formamide enabled separation of Vibrio populations with single nucleotide differences in the amplified fragment. A titration curve for the QC-PCR-DGGE, verified by amending surface water bacterioplankton samples with up to 3 × 10 5 Vibrio cholerae cells, could be approximated by a linear regression of log-transformed values ( R 2 = 0.96). The limit of detection for single populations was 180 cells per extracted sample or about 4 cells per PCR reaction. Environmental samples from the southern Stockholm archipelago in the Baltic Sea and the more saline coastal waters of Skagerrak each carried between 2 and 6 Vibrio populations, and there were major differences between the locations. Notably, multiple Vibrio populations could be detected and quantified against a background of native bacterioplankton exceeding Vibrio population abundance by more than 6 orders of magnitude. Putative identification based on migration in the DGGE gel was verified by parallel cloning and sequencing of PCR products, and representative clones were also characterized by DGGE. This general approach could also be useful for targeting other phylogenetically constrained bacterial groups and assess their abundance and distribution in complex environmental settings.

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.

Aquaporins in endothelia

Aquaporin-1 (AQP1) water channels are expressed widely in microvascular endothelia outside of the central nervous system, including renal vasa recta and tumor microvessels, as well as in non-vascular endothelia in pleura, peritoneum, cornea, and lymphatics. In kidney, AQP1-facilitated water transport in outer medullary descending vasa recta is likely an important component of the urinary concentrating mechanism. However, in most vascular endothelia outside of kidney, it remains uncertain whether AQP1 expression and high water permeability are physiologically important. AQP1 in non-vascular endothelia at the inner corneal surface is involved in the maintenance of corneal transparency. Recently, a new role of AQP1 in endothelial cell migration was discovered in analyzing the cause of defective tumor angiogenesis in AQP1-deficient mice. AQP1 facilitates endothelial cell migration by a mechanism that may involve facilitated water transport across cell protrusions (lamellipodia). AQP1 inhibitors may thus have aquaretic and antiangiogenic activity.

Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis

Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6× His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8 M urea and the recombinant protein was purified by Ni–NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Delta65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H(4)B) and is the site for NO synthesis. The role of H(4)B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H(4)B) with the bacterially expressed Delta65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Delta65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Delta65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H(4)B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H(4)B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain.

A Combined Refolding Technique for Recombinant Human Interferon-γ Inclusion Bodies by Ion-exchange Chromatography with a Urea Gradient

A refolding strategy was described for on-column refolding of recombinant human interferon-γ (rhIFN-γ) inclusion bodies by ion-exchange chromatography (IEC). The rhIFN-γ was expressed in E. colias inclusion bodies. Triton X-100 was used first to wash the rhIFN-γ inclusion bodies before chromatographic refolding. The refolding process was performed by gradually decreasing the concentration of urea in the column after the denatured rhIFN-γ protein had bound onto the ion-exchange gel SP-Sepharose Fast Flow. The refolding and purification process for the denatured rhIFN-γ was carried through simultaneously and the purity of the refolded rhIFN-γ was up to 95%. The effects of protein loading, flow rate, urea gradient length and final urea concentration on the refolding were investigated in detail. Under the optimum conditions, the specific activity of rhIFN-γ was up to 7.5 × 105 IU mg−1and active protein recovery was up to 54%.

The Renaturation of Procarboxypeptidase B by Urea Gradient Gel Filtration and Some Properties of Recombinant Carboxypeptidase B

A new pro-carboxypeptidase (pCPB) gene was cloned by RT-PCR from SD rat pancreas and its overexpression in Escherichia coli resulted in the formation of inclusion bodies (IBs). The IBs of pCPB were solubilized in 8 M urea and successively refolded by urea gradient gel filtration. Subsequently, the renatured pCPB was digested by trypsin. Recombinant active CPB was obtained by passing through DEAE-FF ion exchange and Sephadex-G100 chromatographic column. Capillary electrophoresis assay showed that the purity of the recombinant CPB (rCPB) exceeded 90%. Further, some properties of rCPB were characterized. The optimum of activity was achieved at pH 7-9. The activity of rCPB was inhibited by typical metal chelating agents (EDTA) and Hg2+, and was activated by Co2+ and heat treatment at 40 degrees C. The two-dimension electrophoresis map of rCPB showed that the pI value of rCPB was 5.35. UV absorbance spectrum of the enzyme showed that an absorbance maximum was at 277 nm.