Isolation and purification of biopolymers by affinity chromatography. VI. Preparation and properties of an affinity adsorbent with a polysaccharide spacer for the purification of proteolytic enzymes

A new affinity sorbent has been synthesized — soybean trypsin inhibitor (STI)-amylopectin-hydrazidosuccinyl-Sepharose — and its properties have been studied in comparison with those of an analogous adsorbent without the spacer STI-Sepharose. The STI-amylopectin-hydrazidosuccinyl-Sepharose adsorbent has been used for the purification of trypsin from porcine pancreas and of callicrein from human blood plasma.For Communication V, see [1].Institute of Biological and Medicinal Chemistry, Academy of Medical Sciences of the USSR, Moscow. I. M. Sechenov First Moscow Medical Institute. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 556–560, July–August, 1980.     

Membrane affinity chromatography for analysis and purification of biopolymers

Summary Affinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only about 2.5 min for analysis at a flow-rate of 1.0 mL min⁻¹. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 mL min⁻¹ within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid. It can also be used for micro-scale purification of biopolymers.     

Isolation and purification of endo-polygalacturonase by affinity chromatography in a fluidized bed reactor

Endo-polygalacturonase can be purified and isolated from an industrial pectolytic enzyme preparation using alginate as an affinity adsorbent in a fluidThe adsorption and desorption kinetics of the system have been studied. First the adsorption and desorption conditions were optimized with respect to pThe stability of the beads was tested in repeated adsorption/desorption experiments in a batch fluidized bed reactor isolating the enzyme from an indus     

纳米SiO_2亲和吸附剂的制备及性能

采用水热法一步合成了巯基纳米二氧化硅(SiO_2-SH),随后在其表面修饰亚氨基二乙酸基团(-IDA)得到了SiO_2-SH/IDA,利用-SH和-IDA双官能团更多的吸附溶液中的Ni~(2+),从而得到SiO_2-SH/IDA-Ni~(2+)纳米亲和吸附剂.制备的亲和吸附剂可直接用于六聚组氨酸为标签的(His-tagged)融合蛋白的分离纯化.利用TEM、FT-IR、TG、SDS-PAGE等大型仪器表征了样品的形貌、结构及亲和分离能力.结果表明制备的SiO_2-SH/IDANi~(2+)纳米亲和吸附剂平均粒径为60nm,对His-tagged蛋白具有较好的特异性和较低的检测限(约为1.9×10~(-5)mol/L),且该吸附剂再生能力较强,再生3次后对目标蛋白仍具有较好的分离效果.     

Isolation of an urate-binding protein by affinity chromatography

This paper describes an affinity chromatography procedure to purify an urate binding protein from human serum. The specific ligand was 8-amino-2,6-dihydroxypurine bound to Sepharose through the amino group. The specific elution was obtained with an uric acid or allopurinol solution. Electrophoretic analysis of the eluted protein shows a single sharp band with an α₂-globulin mobility. Molecular weight, determined by gel filtration, is approximately 70,000 daltons.     

Immobilized ferredoxins for affinity chromatography of ferredoxin-dependent enzymes.

An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga. The dissociation constants of immobilized ferredoxin from spinach, barley and S. vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively. The order of stability was S. vulcanus greater than barley greater than spinach. The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts. Over 20 polypeptides were detected. Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system.     

Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins

We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Phosphotungstate as a useful eluent for hepatitis-B virus surface antigen purification by heparin-sepharose affinity chromatography.

On the basis of the heparin-like effect of phosphotungstate (PTA), we have shown that it is useful for the purification of hepatitis-B virus surface antigen (HBsAg) using heparin-Sepharose affinity chromatography. HBsAg was eluted with 0.2-0.6 M NaCl. HBsAg was also eluted with PTA at approximately 1 mM, and the HBsAg fraction thus obtained contained fewer impurities than the corresponding fraction eluted with NaCl. Moreover, PTA yielded HBsAg and hepatitis-B virus e-antigen simultaneously. PTA may specifically reduce the affinity of HBsAg for heparin as well as simply competing with heparin for an anion-binding site of HBsAg. Residual PTA in the eluate was easily decomposed by alkalization, which was useful for subsequent studies.     

Preparation of anhydrochymotrypsin diol silica as selective adsorbent for affinity chromatography of peptides with aromatic amino acids at C-termini

Summary Anhydrochymotrypsin (AHC), a catalytically inert derivative of chymotrypsin in which the serine-residue active site has been converted chemically to a dehydroalanine residue, was immobilized on diol silica by activation with trifluoroethanesulfonyl chloride. A AHC-diol-silica column was used for high-performance affinity chromatographic separation of peptides with aromatic amino acids at their C-termini from other peptides. Faster separations were achieved.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

新型镍离子亲和色谱介质的制备及其性能考察

以氯甲基化聚苯乙烯交联微球(氯球)为载体基质、N,N-二甲基甲酰胺(DMF)为溶剂、N,N-二异丙基乙胺(DIEA)催化,将合成的Lys(Boc)-OEt、氯乙酸乙酯连接到微球上。经水解使羧基裸露,与Ni²⁺螯合,制得新型镍离子亲和色谱介质。将其应用于His-Lys-Tyr三肽及Phe-His-Thr三肽纯化研究。以不含组氨酸的六肽作为杂质模型,用所制备的镍离子亲和介质进行分离。结果表明,所制备的镍离子亲和介质对两个含组氨酸的三肽具有很好的亲和作用,并实现了与不含组氨酸六肽的分离。     

High-performance affinity chromatography of a chick lectin on an adsorbent based on hydrophilic polymer gel

A mouse monoclonal antibody (SIA4-5) which reacts with a chick 14K lectin (C14K) was covalently attached to a new support for high-performance affinity chromatography, TSKgel Tresyl-5PW, which is a preactivated, polymer-based particle. The immobilized antibody (SIA4-5-5PW) thus prepared proved to be useful in measuring not only the molecular properties of C14K but also specific interactions of C14K with SIA4-5 and hapten sugars. The C14K preparation was fractionated according to the oligomeric structure and with slight differences in affinity to SIA4-5 although the former was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Application of the method for quantitative analytical purposes was successful.     

Application of poly-L-lysine to purification of leukocyte cathepsin G by affinity chromatography.

Poly-L-lysine with molecular masses of 3.3-290 kDa increased the amidolytic activities of leukocyte elastase and cathepsin G at low concentration, but had little effect on the activities of pancreatic elastase, alpha-chymotrypsin, plasmin and thrombin. Highly purified cathepsin G was obtained from column of EAH Sepharose 4B or Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose (affinity chromatography) by elution with poly-L-lysine solution (0.4 mg/ml, molecular weight (MW.) 290000 or 2.2 mg/ml, MW. 3300). Leukocyte elastase, adsorbed to Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose, was not eluted with poly-L-lysine solution. The amino acid composition of purified cathepsin G has been determined.     

Sequence-specific DNA affinity chromatography: Application of a group-specific adsorbent for the isolation of restriction endonucleases

The use of sequence-specific DNA affinity adsorbents for the isolation of restriction endonucleases EcoRI and SphI to near homogeneity has been reported. However, the high cost of these adsorbents is a limiting factor for their wider application. This paper reports the application of sequence-specific DNA affinity ligands containing recognition sequences for 34 restriction endonucleasesas group-specific ligands in the isolation of restriction endonucleases. Crude samples of six restriction endonucleases, namely BshFI, BamHI, SmaI, SacII, PvuII and SalI, were shown to bind to these adsorbents and could be eluted at different KCl concentrations. High purification factors and recoveries were obtained. Restriction endonuclease BshFI, an isoschizomer of HaeIII, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose chromatography and oligonucleotide- ligand affinity chromatography. The enzyme exists as a monomer with an apparent relative molecular mass of 34 000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion chromatography.     

An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography

The development and characterization of an artificial protein L (PpL) for the affinity purification of antibodies is described. Ligand 8/7, which emerged as the lead from a de novo designed combinatorial library of ligands, inhibits the interaction of PpL with IgG and Fab by competitive ELISA and shows negligible binding to Fc. The ligand 8/7 adsorbent (Ka approximately 10(4) M(-1)) compared well with PpL in binding to immunoglobulins from different classes and sources and, in addition, bound to IgG1 with K and lambda isotypes (92% and 100% of loaded protein) and polyclonal IgG from sheep, cow, goat and chicken. These properties were also reflected in the efficient isolation of immunoglobulins from crude samples.