Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.     

An assessment of nonspecific adsorption to Eudragit S-100 during affinity precipitation

The problem of nonspecific adsorption to the reversibly soluble-insoluble polymers is of considerable importance in the design of an affinity precipitation protocol. It was seen that activation and coupling of the affinity ligand to the polymer changes the nature of the polymer surface in a significant fashion. The results with pure trypsin, partially purified trypsin preparation, and crude protein extract-containing protein inhibitors of trypsin and α-amylase and the reversibly soluble-insoluble polymer Eudragit S-100, show that nonspecific adsorption may not be a severe limitation in such systems.     

Synthesis and characterization of a water-soluble affinity polymer for trypsin purification

A specific ligand bound polymer has been synthesized for the purpose of purification and stabilization of trypsin, an easily autodigestible enzyme. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenzamidine, a strong trypsin inhibitor, and acrylamide in the absence of oxygen. Kinetic studies on the trypsin inhibition revealed that there was a strong binding between this enzyme and the polymer and the mechanism was of a competitive manner with an inhibition constant of 0.6 x 10(-3)M. Such an affinity polymer was also very effective in preventing trypsin from auto-digestion at 4 degrees C.Based on this finding and the principle of cross flow filtration, a new process has been developed for purification of trypsin from a solution containing chymotrypsin. The experimental data indicated that trypsin was bound to the polymer (MW > 10(5)) and remained in the retentate while unbound chymotrypsin was collected in the filtrate. This purification process has a capability of recovering 98% pure trypsin at 90% yield.     

Trypsin purification by affinity binding to small unilamellar liposomes

A novel protein purification process using affinity-ligand-modified liposomes and membrane ultrafiltration is described. The feasibility of the process was tested using trypsin as the model protein and p-aminobenzamidine (PAB) as the affinity ligand for trypsin. The affinity liposomes were prepared by covalently attaching PAB to the surface of small unilamellar liposomes via the hydrophilic spacer arm diglycolic acid. The liposomes were comprised of dimyristoyl phosphatidyl choline, cholesterol, and dimyristoyl phosphatidyl ethanolamine to which the diglycolic acid was attached. The equilibrium binding constant between trypsin and immobilized PAB was shown to be dependent on the PAB density of the liposome surface. Bound trypsin was eluted from the liposomes by the trypsin inhibitor benzamidine. Trypsin was purified from a trypsin/chymotrypsin mixture and from one of its naturally occurring sources, porcine pancreatic extract. A recovery yield from the crude mixture of 68% was obtained with a trypsin purity of 98%. The affinity-modified liposomes were stable in the complex mixture and retained their trypsin binding capacity after multiple adsorption/elution cycles over a 30-day period.     

Affinity thermoprecipitatin: Contribution of the efficiency of ligand-protein interaction and access of the ligand

Conjugates to two thermoprecipitating polymers, poly(N-vinyl caprolactam) and poly(N-isopropylacrylmide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu-iminodiacetic acid, and p-aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100-1000-fold decrease in enzyme-affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10(-7)-10(-8) M. Such strong affinity of low molecular weight ligands that can provide binding constants of 10(-9)-10(-11) M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand-polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particle of the dispersion of thermoprecipitated ligand-polymer precipitate may result in stripping of enzyme molecules from the surface of the particles. (c) 1993 Wiley & Sons, Inc.     

Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand

An ammonium sulphate fraction (20–60%) of bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) was purified by affinity chromatography to give 6.59-fold purity with 81.48% yield. The same ammonium sulphate fraction was also subjected to ion exchange chromatography and was purified 4.28-fold with 75.95% yield. The ion exchange fraction was subjected to gel filtration and the inhibitor was purified to 6.67-fold with 67.36% yield. Further sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the homogeneity of purified amylase/trypsin inhibitor obtained through affinity, ion exchange and gel chromatography. The molecular weight of the inhibitor was found to be 14 kDa. This purified inhibitor was used as affinity ligand for the purification of a commercial preparation of pancreatic amylase.     

Affinity thermoprecipitation of trypsin using soybean trypsin inhibitor conjugated with a thermo-reactive polymer,poly(N-vinyl caprolactam)

The soybean trypsin inhibitor conjugate with a thermo-reactive water soluble polymer, poly(N-vinyl caprolactam), was successfully used for thermally induced affinity precipitation of trypsin. The validity of the developed procedure was proven by a model separation of trypsin from dilute solution containing a large excess of bovine serum albumin.     

Design and study of peptide-ligand affinity chromatography adsorbents: application to the case of trypsin purification from bovine pancreas

The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C-terminal (site P(1)) for trypsin bio-recognition, a Pro or Ala in site P(2), and a Thr or Val in site P(3). Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 mumol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 mumol/g gel. The K(D) values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K(D) values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K(D) 8.7 muM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 muM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 mumol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. (c) 1997 John Wiley & Sons, Inc.     

Anhydrotrypsin: new features in ligand interactions revealed by affinity chromatography and thionine replacement.

Anhydrotrypsin was isolated in high purity from the product of base elimination from phenylmethanesulfonyl-trypsin, by a single operation of affinity chromatography. The adsorbent used for the chromatography was an agarose derivative coupled with peptides containing C-terminal arginine residues. As the affinity of the adsorbent for anhydrotrypsin was high compared with that for trypsin, purification of the enzyme derivative was easily achieved without the prior inactivation of trypsin which had been regenerated during the elimination reaction. Comparative studies of the ligand interaction specificities with anhydrotrypsin and trypsin confirmed the stronger interaction of the former protein with product-type ligands such as Bz-Arg-OH. No marked differences were observed between them in affinities toward substrate-type ligands such as Bz-Arg-NH2. The higher affinity of anhydrotrypsin was found to be limited to product-type ligands of L-configuration, i.e., the protein displayed an ability to discriminate the L-ligand from its optical isomer. THE PKa value for the ionization form of anhydrotrypsin responsible for the interaction with Bz-Arg-OH was estimated to be 7.60+/-0907     

Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

以壳聚糖为载体亲和层析胰蛋白酶

用活化的壳聚糖为载体,鸡卵粘蛋白(CHOM)为配基,制备了胰蛋白酶的亲和吸附剂。采用该吸附剂亲和层析胰酶,所得产物经SDS-PAGE电泳检测,带中只有一条带颜色较深,且与标准胰蛋白酶带位置几乎相同。实验结果表明1 g壳聚糖可以固定60 mg鸡卵粘蛋白,制成的亲和吸附剂可吸附胰蛋白酶的最大量为118 U/g。以壳聚糖为载体的亲和吸附剂制备过程简单、安全。     

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100

An IgG₁ monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.     

Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands.

An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.     

Integrated removal of nucleic acids and recovery of LDH from homogenate of beef heart by affinity precipitation

Lactate dehydrogenase (LDH) was purified from beef heart homogenate by affinity precipitation. The protein purification was integrated with nucleic acid removal and was done by precipitation of nucleic acids by addition of poly(ethylene imine) PEI onto which a ligand, Cibacron blue, had been coupled. The yield of LDH after elution from the precipitate was 63%, the purification factor 6.9 and the nucleic acid content was reduced by 98%. The capacity of the affinity polymer Cibacron blue-PEI is dependent on the nucleic acid concentration in the homogenate. The beef heart homogenate had an unfavourable ratio of nucleic acids to LDH. Precipitation with recirculated Cibacron blue-PEI, already complexed with some nucleic acids, improved the yield of the enzyme to 74%. The loss of Cibacron blue-PEI, when recirculated, was less than 1% after each cycle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Affinity chromatography of trypsin and related enzymes. IV. Quantitative comparison of affinity adsorbents containing various arginine peptides

In order to study the mechanism of substrate binding of trypsin by affinity chromatography, we synthesized various L-arginine-terminated oligopeptides having different chain length and amino acid sequences, and immobilized them on agarose gel. The interaction of beta-trypsin with these adsorbents was studied by a quantitative affinity chromatographic procedure which gave the dissociation constant (Kd) of the trypsin-immobilized ligand complex. This procedure proved to be very useful and to give information equivalent to that obtained by kinetic procedures. The contribution of the amino acid residue at P2 of the ligands to the affinity was studied by using tripeptide (Gly-X-Arg) Sepharoses, and alanine was found to be more effective than glycine or valine. This conclusion was supported by a kinetic experiment in which Ki values of the corresponding soluble tripeptides (Ac-Gly-X-Arg) were determined. A significant decrease in Kd was observed when the ligand was elongated from dipeptide to tripeptide. However, Kd decreased only slightly when the ligand was elongated further. This suggests that a tripeptide is sufficiently long as a ligand. On the basis of these results, the mode of substrate binding of trypsin is discussed.     

固定化胰蛋白酶亲和层析法制备低STI残存大豆分离蛋白质

聚苯乙烯阴离子交换树脂(GM201)经预处理除去杂质后先与戊二醛(2—6％)反应,再与胰蛋白酶(5000u/mg,8—10mg/mL,pH 8.0)反应即制得固定化胰蛋白酶。此法得到的固定化胰蛋白酶具有良好的热稳定性,贮藏稳定性和操作稳定性,可用于工业化目的。脱脂豆粉经萃取(PH9.0)后,稀释4倍,在pH5.0下沉淀分离出大豆球蛋白,然后用酸性水(pH5.0)洗涤两次,并进行碱溶与酸沉淀两次,即可将大豆分离蛋白质的STI残留降低到1.85％,比活性降到1u/mg以下。最后再用固定化胰蛋白酶亲和层析,就可以除去大豆分离蛋白质中残留的STI。     

The development and application of a new affinity partitioning system for enzyme isolation and purification.

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.     

Isolation and separation of α-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation

The concept of immobilized metal affinity chromatography (IMAC) was integrated with affinity precipitation for the single step isolation of -amylase inhibitors I-1 and 1-2 from the seeds of ragi (Indian finger millet, Eleusine coracana). -Amylase inhibitor I-1 was purified 13-fold with a yield of 84%, using Cu(II) loaded thermosensitive metal chelate copolymer of N-isopropylacrylamide (NIPAM) and 1-vinyl imidazole (VI). The protein also showed trypsin inhibitory activity. The binding of the protein to the copolymer was strongly pH dependent. -Amylase inhibitor I-2 was recovered in the supernatant as unprecipitated protein with significant purification and constituted 27% of the total inhibitor power. The yield with respect to inhibitor I-2 was around 85%. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed significant purification of inhibitor I-1 and indicated evident separation of the two proteins on metal chelate affinity precipitation.     

Affinophoresis of trypsins

We devised a new separation technique for the protein, "affinophoresis," which is based on its specific affinity and utilizes electrophoresis. This technique requires a carrier macromolecule, "affinophore," which contains both an affinity ligand for a certain protein and many charges, either positive or negative, in order to migrate rapidly in an electric field. When a mixture of proteins is electrophoresed in the presence of the affinophore, the protein having an affinity with the ligand will form a complex with the affinophore. This results in a change in the apparent electrophoretic mobility. If the protein is sufficiently accelerated, we can separate it from other materials. A cationic affinophore for trypsin was prepared. Soluble dextran MW approximately 10,000) was coupled with a DEAE-group and m-aminobenzamidine, a competitive inhibitor of trypsins. Electrophoresis of trypsins from several origins on agarose gel plates in the presence of the affinophore showed that affinophoresis actually occurred. The electrophoretic mobilities of trypsins increased towards the cathode, the same direction as the affinophore movement. The presence of leupeptin and treatment of the trypsins with TLCK suppressed the effect of the affinophore. Streptomyces griseus trypsin, contained in Pronase, was easily separated and detected. This procedure is distinct from affinity chromatography and so-called affinity electrophoresis in that the support of the affinity ligand moves, and has advantages especially for analytical purposes: for example, the detection of specific molecules regardless of their isoelectric points.     

Water-soluble nonionic surfactants for affinity bioseparations

Reversible competitive inhibitors of the three enzymes beta-galactosidase, trypsin, and serum cholinesterase have been covalently attached to nonionic ethoxylated surfactants. The binding of the resulting affinity-derivatized surfactants to the respective enzymes has been quantified by measuring Michaelis-Menten inhibition constants with kinetic assays. The surfactant-inhibitor of serum cholinesterase, octaethylene glycol monohexadecy ether pyridinium (C(16)E(8)-PYR), was adsorbed in aqueous solution to an octadecyl-bonded reverse-phase silica packing in a 2 x 0.2 cm stainless steel test column. The ability of the test column to function as a high-performance affinity chromatography (HPAC) column was determined by applying a mixture of bovine serum albumin and cholinesterase (4:1 w/w). Virtually all of the cholinesterase bound and was eluted by applying a gradient in ionic strength. The applied cholinfesterase was recovered with a yield of over 90% and an 11-fold purification. An aliquot of raw horse serum was then purified in the same fashion with a yield of 84% and a 280-fold purification. The surfactant-inhibitor was easily removed from the column with an alcohol wash for sterilization, cleaning, or application of a different affinity ligand. Moreover, the ligand density on the column can be easily manipulated by adsorbing mixtures of derivatized and underivatized surfactants. Leakage of ligands from the support seems to be minimal since the cholinesterase affinity column was operated efficiently after being exposed to 24,000 column volumes of buffer. The application of this technique to high-capacity, high-throughput reversible affinity purifications is limited only by the ability to identify suitable ligands.