A study on the selective binding of apoprotein B- and E-containing human plasma lipoproteins to immobilized rat serum phosphorylcholine-binding protein

Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism.     

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography

Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap? 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose? 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose? 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.     

Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti (4+)-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti (4+) through the chelating interaction between phosphate groups on the polymer and Ti (4+). The resulting Ti (4+)-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti (4+)-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti (4+)-IMAC to other enrichment methods including Fe (3+)-IMAC, Zr (4+)-IMAC, TiO 2 and ZrO 2, and demonstrate superior selectivity and efficiency of Ti (4+)-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti (4+)-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.     

Protein dye affinity chromatography using immobilized tetraiodofluorescein

The chromatographic behavior of a heterogeneous protein mixture and of a series of homogeneous proteins on the immobilized dye tetraiodofluorescein has been observed and analyzed. Less than 6%, of the millimolar concentration of dye immobilized to a porous agarose matrix is accessible to protein. The affinity of a protein for immobilized dye is dramatically increased by insertion of apolar spacer atoms between the dye and the matrix. Dye columns constructed with a 9-atom spacer can be used to advantage for the retention and competitive elution of proteins not found previously amenable to dye chromatography.     

Removal of autoantibodies by 4-mercaptoethylpyridine-based adsorbent

Extracorporeal immunoadsorption (ECI) therapy using Staphylococcal Protein A columns has proven effective for removing autoantibodies and circulating immune complexes from patients selectively, providing a promising treatment for autoimmune diseases. However, due to the drawbacks of Protein A in terms of cost and stability, the widespread use of Protein A based ECI is limited. In this study, we investigated the feasibility of 4-mercaptoethylpyridine (MEP, MW 139 Da), a simple and inexpensive synthetic compound, as an alternative to Protein A for autoantibody removal therapy. MEP-based adsorbents were prepared by coupling MEP to Sepharose CL-6B. We found that ligand density was an adjustable parameter for the synthesis of adsorbents aiming at different pathogenic factors, depending on the class of antibody. MEP-Sepharose with a ligand density of 98.8 μmol/ml could remove 80% of the anti-double-stranded DNA antibodies from human serum, whereas a ligand density of 64.5 μmol/ml was enough to remove 96% of the rheumatoid factor (RF) in the serum. Moreover, MEP-based adsorbents showed a lower degree of individual differences compared to Protein A-Sepharose. RF removal of 90% was achieved for all 12 serum samples from different individuals. Among the 14 serum samples derived from systemic lupus erythematosus patients, 11 samples had markedly reduced antinuclear antibody titers. In addition, non-specific adsorption of plasma components to MEP-Sepharose was limited, and the binding capacity of the absorbent for IgG was still about 20 mg/ml of gel after 10 cycles. The results indicated that MEP-based adsorbent could offer a new type of adsorber for the treatment of autoimmune diseases.     

Synthesis and secretion of serum phosphorylcholine-binding protein by rat hepatocytes

The incorporation of [1-14C]glucosamine into rat serum phosphorylcholine-binding protein in an isolated rat hepatocyte system was used to demonstrate the synthesis and secretion of this protein by the liver. The hepatocytes after incubating with colchicine resulted in an increased intracellular accumulation of phosphorylcholine-binding protein and less of the synthesized phosphorylcholine-binding protein was secreted into the medium. The synthesis of phosphorylcholine-binding protein was found to be significantly impaired when the hepatocytes were incubated with tunicamycin. The radiolabelled phosphorylcholine-binding protein co-eluted with exogenous phosphorylcholine-binding protein as a homogeneous peak by affinity chromatography. The identity of the radiolabelled phosphorylcholine-binding protein was further established by quantitative immunoprecipitation, polyacrylamide gel electrophoresis and isoelectric focusing.     

The continuous isolation of trypsin by affinity adsorbent recycling

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 mug of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.     

Removal of pentachlorophenol by immobilized horseradish peroxidase

Researches on the polymerization of aqueous pentachlorophenol (PCP) by the catalysis of horseradish peroxidase (HRP) with the existence of hydrogen peroxide (H₂O₂) were conducted. Factors, such as acidity, temperature, enzyme activity, and initial concentration of PCP and H₂O₂ that could influence the degradation were studied. Results showed that the optimum pH value for free enzyme was 5–6; relative higher temperature could accelerate the reaction greatly; PCP removal increased with an increase of enzyme concentration, and PCP (initial concentration 12.6 mg/L) removal percentage could reach nearly 70% under the highest enzyme concentration (about 0.05 u/ml) adopted in the experiment; removal percentage increased slightly with an increase of initial concentration of PCP, and when initial PCP concentrations were 13.0 and 0.7 mg/L, the removal percentages were about 73.7% and 35.7%, respectively; the molar ratio of the reaction between PCP and H₂O₂ was about 1:2.Based on the above results, researches on the removal of PCP by the immobilized HRP were conducted. The free HRP was immobilized on the polyacrylamide gel prepared by gamma-ray radiation method; then the immobilized HRP was filled into a column, and PCP was successfully removed by the immobilized HRP column. The results were compared with results using free HRP enzyme, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP, and when pH=5.15, the immobilized HRP could reduce PCP with initial concentration 13.4 mg/L to the concentration of 4.9 mg/L within 1 h, and the immobilized HRP column could be used to repeatedly.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

LdARL-1 His-tagged recombinant protein: purification by immobilized metal affinity expanded bed adsorption

Previously we have cloned three ADP-ribosylation factor-like (ARL) genes from the parasitic protozoan Leishmania donovani: LdARL-3A and 3B, LdARL-1. LdARL-3A was previously purified as an active native form, which was able to bind GTP in vitro. In this paper, we have performed the production and the purification of Histidine-tagged (His-tagged) LdARL-1 recombinant protein by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology. This protein was purified with more than 95% purity and could be successfully used for GTP-binding assay.     

Protein fractionation using fast flow immobilized metal chelate affinity membranes

A new group-specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified "active surfaces" were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu-IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.The results presented here represent the development of a new fast flow affinity membrane process-immobilized metal affinity membranes (IMAM). (c) 1994 John Wiley & Sons, Inc.     

Suitability of immobilized metal affinity chromatography for protein purification from canola

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.     

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Design of affinity tags for one-step protein purification from immobilized zinc columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.     

Removal of methylene blue dye from aqueous solutions by adsorption using yellow passion fruit peel as adsorbent

The removal of color from aquatic systems caused by presence of synthetic dyes is extremely important from the environmental viewpoint because most of these dyes are toxic, mutagenic and carcinogenic. In this present study, the yellow passion fruit (Passiflora edulis Sims. f. flavicarpa Degener) peel a powdered solid waste, was tested as an alternative low-cost adsorbent for the removal of a basic dye, methylene blue (MB), from aqueous solutions. Adsorption of MB onto this natural adsorbent was studied by batch adsorption isotherms at room temperature. The effects of shaking time and pH on adsorption capacity were studied. An alkaline pH was favorable for the adsorption of MB. The contact time required to obtain the maximum adsorption was 56 h at 25 degrees C. Yellow passion fruit peel may be used as an alternative adsorbent to remove MB from aqueous solutions.     

Purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography

Over the past 10 years, the baculovirus-insect cell system has become a powerful and versatile tool for the expression of a variety of heterologous proteins. In order to simplify separation of a cloned protein from the baculovirus-insect expression system, we have cloned a gene encoding for the protein of interest, a structural protein (VP2) of a strain (E/DEL) of infectious bursal disease virus (IBDV), with a metal ion binding site (His)(5) at its C-terminus. This chimeric protein (VP2H) has been expressed and one-step affinity purified with immobilized metal ions (Ni(+2)). With antigen capture-enzyme-linked immunosorbent assay (AC-ELISA), we determined that the conformation of this chimeric protein was no different from the recombinant wild-type VP2 protein. However, the two proteins (VP2 and VP2H) can be distinguished and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected immunologically following Western blotting. (c) 1994 John Wiley & Sons, Inc.     

The kinetics of binding of serum lipoproteins by immobilized heparin.

A model arterial system of heparin immobilized on an agarose gel was used to study the amount and kinetics of binding of porcine serum lipoproteins to heparin. Binding occurred to lipoproteins in the density range 1.006 less than d less than 1.062, but there was no binding with high density lipoprotein. A theoretical model of the kinetic experiments was formulated and used to demonstrate that the rate of the binding reaction could be considered instantaneous relative to the rate of transport of lipoproteins. Extrapolation of these results to arterial levels of glycosaminoglycans and lipoprotein indicate that complexes of lipoprotein and the glycosaminoglycans could account for much of the cholesterol entrapment in atherosclerotic lesions.     

Removal of chlorophenols from wastewater by immobilized horseradish peroxidase

Immobilization of horseradish peroxidase on magnetite and removal of chlorophenols using immobilized enzyme were investigated. Immobilization by physical adsorption on magnetite was much more effective than that by the crosslinking method, and the enzyme was found to be immobilized at 100% of retained activity. In addition, it was discovered that horseradish peroxidase was selectively adsorbed on magnetite, and the immobilization resulted in a 20-fold purification rate for crude enzyme. When immobilized peroxidase was used to treat a solution containing various chlorophenols, p-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, each chlorophenol was almost 100% removed, and also the removal of total organic carbon (TOC) and adsorbable organic halogen (AOX) reached more than 90%, respectively. However, in the case of soluble peroxidase, complete removal of each chlorophenol could not be attained, and in particular, the removal of 2,4,5-trichlorophenol was the lowest, with a removal rate of only 36%. (c) 1996 John Wiley & Sons, Inc.     

Removal of inorganic ions from wastewaters by immobilized microalgae

Anabaena doliolum and Chlorella vulgaris immobilized on chitosan were more efficient at removing NO₃ ^–, NO₂ ^p–, PO₄ ^3– and CR₂O₇ ^2– from wastewaters than cells immobilized on agar, alginate, carrageenan or even free cells. Carrageenan-immobilized cells, however, were better at removing NH₄ ⁺ and Ni²⁺. The PO₄ ^3– uptake capacity was significantly increased in cells starved of PO₄ ^3– for 24 h. Agar-immobilized cells, though having good metal and nutrient uptake efficiency, had only a slow growth rate. Chitosan is recommended as an algal support for wastewater detoxification.The authors are with the Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, Varanasi-221005, India