Synthesis of thermo-sensitive copolymer with affinity butyl ligand and its application in lipase purification

In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer P_NNB was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28 °C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer P_NNB and butyl ligand density was 80 μmol g^?1 polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35 °C adsorption temperature, 120 min adsorption time and 0.5 mg ml^?1 initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q_m) of the affinity polymer for lipase was 24.8 mg g^?1polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14 IU mg^?1 to 506 IU mg^?1 protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution.     

Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

Development and application of thermo-sensitive magnetic immunomicrospheres for antibody purification

Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum.Correspondence to: A. Kondo     

Preparation of a light-sensitive and reversible dissolution copolymer and its application in lysozyme purification

A novel light-sensitive and cation-exchange copolymer (PNBCC) has been synthesized by random copolymerization of chlorophyllin sodium copper salt, crylic acid, n-butyl acrylate, and N-isopropylacrylamide. The PNBCC copolymer showed reversible dissolution and could be precipitated by 488 nm laser irradiation with the least light density of 1.70 x 10(5) W/m(2). By optimizing the ratio of monomers, pH, and ion concentration, over 95% copolymer was recovered by laser irradiation. The copolymer was used to purify lysozyme as light-sensitive cation exchanger, and its adsorption matched a Langmuir adsorption isotherm with maximum adsorption capacity of 98,900 U/g and dissociated constant of 852 U/mL. By applying the copolymer to the separation of lysozyme from egg white, the specific activity of lysozyme was improved from 399 to 6346 U/mg and the recovery of lysozyme achieved 81.3%.     

Copper affinity precipitation as an initial step in protein purification

Summary The affinity of copper ions for histidine residues can be utilized to carry out fractional precipitation of proteins. Using this approach it is possible to purify concanavalin A by a simple two step procedure. The fold purification obtained was 4.5 fold.     

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.     

A novel matrix for high performance affinity chromatography and its application in the purification of antithrombin III

Viscose fiber, a regenerated cellulose, was evaluated for using as a novel matrix for high performance affinity chromatography. With a one-step activation with epichlorohydrin, heparin can be readily covalently attached to the matrix. This heparin-viscose fiber material was used for purifying antithrombin III (AT III) from human plasma. The purity of the AT III from this one-step purification is 93% as measured by SDS-PAGE and the protein recovery yield is about 90%. This column is highly specific as described by the dissociation constant of the complex of immobilized heparin and AT III, which was 2.83 x 10(-5)mol/L. And more important, this viscose fiber material demonstrated its excellent mechanical property that allows the flow rate to reach up to 900 cm/h or more.     

Binding of calcium to lysozyme and its derivatives

Bindings of calcium to lysozyme and its derivatives were studied by UV difference spectroscopy at various pH's. The binding constant was ca. 40 m-1 at around neutral pH. The binding caused proton release from lysozyme and did not inhibit the binding of tri-N-acetylglucosamine to lysozyme. In the presence of 0.2 M Ca2+, lysozyme showed 26% of the activity of the free enzyme toward hexa-N-acetylglucosamine but the cleavage pattern was similar to that of the free enzyme. Thus, calcium was predicted to bind near the catalytic carboxyls to cause inhibition of lysozyme activity. It was found from the results of protease digestion that calcium binding shifted the native-denatured transition in lysozyme toward the native state.     

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.     

Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a K_D of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.     

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate

β-amylases are used in production of maltose syrup. It is shown that sweet potato β-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of β-amylases.     

Synthesis and NMDA-receptor affinity of 4-oxo-dexoxadrol derivatives

A synthesis of novel dexoxadrol analogues is described, which allows modifications of the piperidine substructure. The key step of the synthesis is a hetero Diels-Alder reaction of the imine 12 with Danishefsky's diene 6. After separation of the diastereomeric piperidones 14a and 14b, the relative configuration of the unlike configured piperidone 15b was determined by X-ray crystal structure analysis. In receptor binding studies the like configured secondary amine 15a (racemate) showed considerable affinity toward the phencyclidine binding site of the NMDA receptor (Ki=470 nM).     

Synthesis of a disulfide affinity adsorbent for purification of estrogen receptor

Synthesis of an estrogen affinity adsorbent containing a disulfide linkage between the steroid and stationary matrix permitted facile purification of high affinity estrogen binding proteins. Following affinity chromatography of either antibody directed against estrone 17-carboxymethyloxime — bovine serum albumin or immature calf uterine cytoplasmic estrogen receptor proteins, the specifically bound protein was recovered by incubating the adsorbent with 2-mercaptoethanol. Crude antibody and uterine cytosol was prepared for affinity chromatography in buffer containing 10^?3 to 10^?2M cystamine (S-S) to block SH-containing proteins, in order to protect the adsorbent against protein-mediated S-S ag SH exchange. Cystamine was found to markedly stabilize crude cytosol receptor protein by 200–300% compared with preparations obtained under ordinary conditions. Disulfide affinity adsorbents are versatile in that they can be used either under conventional conditions of specific protein recovery, or with 2-mercaptoethanol which removes the ligand and bound protein from the stationary matrix quantitatively.     

Studies on lectins. XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.     

Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b

The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.