Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.     

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.     

Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide

Polycystic ovary syndrome is a very common disease affecting 6.5% of women. It is frequently associated with obesity with the link between the two disorders being insulin resistance. From morbidly obese women submitted to surgical treatment of obesity, we obtain intra-abdominal adipose tissue samples in order to compare protein patterns between women with and without polycystic ovary syndrome by two-dimensional gel electrophoresis (2-DE) analysis. Adipose tissue is a complex material extremely rich in lipids. To improve protein solubilization a lysis buffer containing 8.4 M urea, 2.4 M thiourea, 5% 3-((3-cholamidoproyl)dimethyl-amino)-1-propanesulfonate and 50 mM dithiothreitol was used and samples were centrifuged to remove fat. Hydroxyethyl disulfide was added to increase resolution in the alkaline region (Olsson, I., Larsson, K., Palmgren, R., Bjellqvist, B., Proteomics 2002, 2, 1630-1632) and it also improved resolution in a wide pH range (3-10). Our work shows for the first time 2-DE maps of human adipose tissue and identifies some of the proteins by mass spectrometry. This information will aid studies on metabolic diseases such as polycystic ovary syndrome, obesity, hypertension and type 2 diabetes.     

Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis

One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.     

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.     

Purification of the bovine rod outer segment Na+/Ca2+ exchanger

Optimal conditions for solubilization and stabilization of the Na+/Ca2+ exchanger from rod outer segments were examined. The exchanger was found to be most stable at low detergent concentrations (7.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), greater than or equal to 100 mM NaCl, pH 7.0-7.5, and with 0.1% added soybean asolectin. The sulfhydryl-modifying reagent, dithiothreitol, caused a loss of exchanger activity and was omitted throughout the purification procedure. These conditions were used to purify the Na+/Ca2+ exchanger from rod outer segments by a combination of selective solubilization, ion exchange, and wheat germ agglutinin chromatography. The procedure achieves a 336-fold increase in exchanger specific activity. The presence of exchanger activity most closely correlates with a polypeptide of molecular mass 215-kDa. Exchanger activity in both the crude rod outer segments and the purified exchanger is specifically dependent upon the presence of K+ in the assay medium; neither choline nor Li+ can substitute for K+.     

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.     

Comparison of the B- and Z-form hairpin loop structures formed by d(CG)5T4(CG)5

The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.     

Solubilization of nuclear steroid 5 alpha-reductase from rat ventral prostate

delta 4-Steroid-5 alpha-reductase (3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.22), is a membrane-bound enzyme. In the ventral prostate of the rat, its activity is found within the nuclear envelope. Solubilization of this enzyme can only be achieved in the presence of detergents. We studied the inhibitory effect of various detergents on 5 alpha-reductase activity as a function of detergent concentration, of pH, of incubation time, of salt concentration and of additives to the buffer system. Four detergents (Lubrol WX, CHAPS, L-alpha-lysophosphatidylcholine and octyl D-glucopyranoside) were selected for subsequent solubilization studies. The overall recovery of solubilized enzyme activity was about 30% when compared to 100% of 5 alpha-reductase activity found in freshly prepared nuclei. Up to 20-30% of the nuclear proteins were extracted during the solubilization procedure. Among the various treatments tested, a concentration of 3 mg/ml L-alpha-lysophosphatidylcholine per 10 mg/ml of nuclear protein in the presence of 5 mM MgCl2, 0.1 M KCl, 0.1 M sodium citrate and 5 mM NADPH yielded the maximal enzymic activity of 56%, 15% of the nuclear proteins being solubilized in an active and stable form. The activity in these extracts could be kept stable for 2 days at 4 degrees C with a recovery of 75% of enzymic activity. A 3-fold increase of specific 5 alpha-reductase activity was obtained during solubilization under optimal conditions.     

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.     

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis.

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14.     

Two-dimensional mapping as a tool for classification of green coffee bean species

Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two-dimensional (2-D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and beta-mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 microL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3-(3-cholamidopropyldimethyl-amino)-1-propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2-D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up-regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down-regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix-assisted laser desorption/ionisation-time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase-high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Effect of insulin to decrease glucose transport in dissociated cells from the R3230AC mammary adenocarcinoma of diabetic rats.

Dissociated cells of the R3230AC mammary tumor were found to take up glucose by diffusion and by a passive carrier system. Using labeled 3-O-methylglucose as the probe, the following properties of the passive carrier were identified: (1) specificity for glucose, (2) competition by galactose and mannose but not by mannitol and fructose, (3) inhibition by phloretin but not by phloridzin, (4) temperature sensitivity, and (5) a Km for transport of 3-4 mM. The effects of insulin in vitro on carrier-mediated glucose transport were investigated in tumor cells from diabetic rats. At 10-9 M insulin, a time-related decrease in v for transport was observed resulting in an increased calculated Km (2- to 3-fold increase after 60-90 min incubation with insulin); only slight effects on V were obtained. This unusual response in v to insulin was observed when glucose was present in the medium at 2 mM and 5 mM, but not at 20 mM glucose. The effect of insulin to decrease the v was dose-related, with the major effects seen between 10-10M and 10-8M. The apparent decrease in glucose entry in vitro may in part explain the ability of insulin to inhibit growth of this tumor in vivo.     

Comparison of variability associated with sample preparation in two-dimensional gel electrophoresis of cardiac tissue.

Variability is a major complicating factor in analysis by two-dimensional gel electrophoresis. Improvements in methodologies have focused on improving individual gel quality rather than reproducibility. We homogenized rat cardiac tissue and rehydrated using a matrix of buffers to determine the optimal sample conditions. Six buffers were used to solubilize the proteins. Solubilized proteins were separated by isoelectric focusing using four buffers. Gels were run in triplicate to assess the method of preparation yielding the least variability. Number of spots and variability were different between conditions. Proteins solubilized in a buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, ampholytes, DTT, and protease inhibitors and focused in a buffer containing 9 M urea and 4% NP40 had the lowest coefficient of variation. Variability was compared across isoelectric point ranges and was different. Minimizing technical variability in two-dimensional polyacrylamide gel electrophoresis is critical to identify differences between conditions. Sample preparation should be optimized to minimize variability as well as to maximize the number of spots seen.     

Capillary electrophoresis screening of poisonous anions extracted from biological samples

A method was developed for screening human biological samples for poisonous anions using capillary electrophoresis (CE) employing indirect UV detection. The run buffer consisted of 2.25 mM pyromellitic acid, 1.6 mM triethanolamine, 0.75 mM hexamethonium hydroxide and 6.5mM NaOH at pH 7.7. Biological samples were pretreated using solid phase extraction. The method was applied to the analysis of human blood, plasma, urine, and intestinal contents. Twenty-nine different anions were detectable at aqueous concentrations of 1 part per million (ppm) with a typical analysis time less than 20 min. Intraday migration time R.S.D. and peak area R.S.D. for blood samples were less than 1.1% and 6.3%, respectively. Interday migration time R.S.D. for plasma samples ranged from 7.5% to 10.4%. The new method produced efficient separations of various target anions extracted from complex biological matrices.     

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.     

Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant streptokinase from E. coli inclusion bodies

The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD(600)=40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l(-1) was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 x 10(5) in cell lysate to 0.53 x 10(5)IU mg(-1) resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96 x 10(5)IU mg(-1)) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.