The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera

The Kdp system from Escherichia coli is a derepressible high-affinity K+-uptake ATPase. Its membrane-bound ATPase activity was approximately 50 mumol g-1 min-1. The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) and N-ethylmaleimide (Ki = 0.1 mM). The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate. Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS. Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme. In contrast, the anti-KdpC serum did not inhibit enzyme activity.     

Affinity purification of serine proteinase from Deinagkistrodon acutus venom

An affinity protocol was developed for the preparation of the main serine proteinase from Deinagkistrodon acutus venom on industrial scales. As affinity ligand, l-arginine was composed to medium and its structure was confirmed by ESI-MS analysis. The purification process consisted of one major affinity chromatography step to remove more than 95% of other proteins, and a polishing step of DEAE ion-exchange chromatography for removal of minor contaminants. The serine proteinase was 100% pure analyzed on HPLC Vydac C4 column, 99.4% on TSK G3000SW column, and 97.7% with SDS-PAGE analysis. The yield of the main serine proteinase was 3.6% of crude venom protein, the recoveries of typical fibrinogen (Fg) clotting activity and arginine esterase activity of serine proteinase were 82.2% and 84%, higher than those of other reported traditional protocols, the proteinase also showed arginine amidase activity. Reducing SDS-PAGE analysis showed that the arginine esterase was a single polypeptide with the mass of approximately 40 kDa, while MALDI-TOF-TOF-MS analysis showed that the purified proteinase should be a approximately 34 kDa glycoprotein. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 9.93 x 10(-5) and 38.1mg/g medium in absorption analysis.     

Affinity purification of a cholesterol oxidase expressed in Escherichia coli

A cholesterol oxidase (COD) gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), an affinity protocol was developed for the preparation, and industrial application of this method was of great potential. Riboflavin was chosen as the affinity ligand, and it was coupled with Sepharose 4B through some spacers. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture the target protein. The purified cholesterol oxidase was 99.5% pure analyzed on HPLC Vydac C4 column, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 9.8% of crude extracted proteins; the recovery of typical cholesterol oxidase activity was 90.1%, higher than that of other reported traditional protocols. Reducing SDS-PAGE analysis showed that the enzyme was a single polypeptide with the mass of ～50 kDa. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 1.0 μg/g medium and 74.5 mg/g medium in absorption analysis. K(m) and V(max) of cholesterol oxidase activity for the purified enzyme were 25.5 μM and 16.4 μmol/(min mg), respectively.     

Purification of subtilisin by single-step affinity chromatography

The dye 4-(4-aminophenylazo)phenylarsonic acid was coupled to activated CH-Sepharose 4B and the resulting affinity matrix was shown to be highly efficient for the purification of subtilisin. A crystalline subtilisin was purified to homogeneity using this affinity chromatography procedure with a purification fold of 1.4 and with an enzyme activity yield of 98%. Similarly subtilisin from a crude enzyme preparation was purified to 211 fold by this single step procedure with 94% recovery of the enzyme activity. The purified enzymes were shown to be homogeneous by polyacrylamide gel electrophoresis.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase

Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the K(m) value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase.     

An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions.     

Purification of beta-glucocerebrosidase by preparative-scale high-performance liquid chromatography: the use of ethylene glycol-containing buffers for chromatography of hydrophobic glycoprotein enzymes

beta-Glucocerebrosidase, partially purified by the method of F. S. Furbish et al. (1977, Proc. Natl. Acad. Sci. USA 74, 3560-3563), was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to contain, in addition to the desired enzyme, variable amounts of a very hydrophobic contaminant (apparent Mr 45,000). Purification of the enzyme was accomplished by gel-permeation HPLC on a TSK 3000 SW column (0.7 X 60 cm). Adsorptive losses of protein on the column were minimized by using buffers containing up to 50% ethylene glycol. We have examined the effects of varying the ethylene glycol concentration on the elution times and recoveries of the two major proteins in this preparation. The high reproducibility of the individual chromatograms permitted the use of an automatic sampler and fraction collector to perform multiple, continuous runs for the purification of milligram quantities of enzyme. Multiple runs of a preparative-scale column, TSK G3000 SWG (2.15 X 60 cm), permitted gram-scale purification of beta-glucocerebrosidase without loss in efficiency of separation. Recovery of enzyme activity is greater than 94% with less than 1% contamination by other proteins. Reaction of enzyme prepared in this fashion with rabbit polyclonal antiserum or mouse monoclonal anti-beta-glucocerebrosidase shows the enzyme to be pure and not immunologically related to the 45,000 Mr contaminant. The specific activity of enzyme prepared by this means is 1.6 X 10(6) nmol/h/mg protein. Inclusion of ethylene glycol in buffers was shown to overcome hydrophobic protein interactions with TSK 3000 SW column matrices for both the soluble human lysosomal enzyme alpha-galactosidase A and the plant toxin ricin.     

A method of isolation of partially purified alkaline peptide hydrolase from Drosophila melanogaster larvae

A semipreparative method is developed for preparing peptidohydrolase from Drosophila melanogaster larvae which involves the stages of extraction, salting-out, gel-filtration and ion-exchange chromatography. It is established that the maximal (up to 81%) yield of the enzyme is observed with the single extraction in the alkaline medium. The main bulk of the enzyme is salted-out in the low acid 3 M ammonium sulphate solution. Gel-filtration on column with Sephadex-25 provides complete salting-out of the enzyme-containing fraction, and ion exchange chromatography on CM-cellulose--a considerable purification of the enzyme under study. A degree of the obtained purification of the enzyme under study. A degree of the obtained peptidohydrolase preparation purity in acid and alkaline medium is determined by the method of electrophoresis in PAAG. At all stages of the preparation the enzyme possesses the casein-lytic activity and is able of hydrolyzing the ethyl ester and benzoyl arginine p-nitroanilide.     

A new method for the purification of RNA polymerase II (or B) from the lower eukaryote Physarum polycephalum. The presence of subforms

The DNA-dependent RNA polymerase II or B from the lower eukaryote Physarum polycephalum has been purified to apparent homogeneity by a new method employing poly(ethylene imine) precipitation and elution, and heparin-Sepharose affinity chromatography. The method is readily scaled up or down and affords a purification of over 5000-fold with a yield of 35-45%. The procedure is easy to perform and can be carried out in less than three days even on a large scale. Furthermore, it gives enzyme of higher purity and in at least 10-fold greater yield than previously published procedures for its purification from this organism. These improvements have allowed the detection of a series of subforms of the enzyme. The combination of precipitation using poly(ethylene imine) with chromatography on heparin-Sepharose may prove useful in the preparation of other proteins which interact with nucleic acids.     

Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds

Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min.     

A highly efficient preparative-scale generic immunoaffinity chromatography method for the purification of antibodies to hydrophobic haptens: purification procedure and monitoring tests

A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50-150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS-PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.     

Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method

Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.     

Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (greater than 95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.     

Overproduction and characterization of xylanase B from Aspergillus niger

The xynB gene, which encodes endo-beta-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg x L(-1). The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 degrees C with stability in the pH range 4.0-7.0 and temperature up to 50 degrees C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg x mL(-1) and 3881 U x mg(-1), respectively.     

Flexprep: Scale-flexible rapid plasmid preparation for analysis of recombinant clones

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones. this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3–10 kbp plasmid vectors in <10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.     

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Purification of histidine-tagged T4 RNA ligase from E. coli

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.     

Human renal renin. Complete purification and characterization

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.     

Purification of rat hepatic glucokinase

We have developed a rapid, reliable procedure for the purification of rat hepatic glucokinase. The purification utilizes DEAE-cellulose, two affinity chromatography steps, and high-performance liquid chromatography. Glucokinase with a specific activity of 240 units/mg, a 42 K-fold purification, and a yield of 60% is obtained. The enzyme appears as a homogeneous band, with over 99% purity as assessed by polyacrylamide gel electrophoresis. The purification procedure can be completed in 5 days.