Rapid purification of beta-glucuronidase from human liver by immunoaffinity chromatography employing specific murine monoclonal IgG binding to tresyl-activated agarose.

We have developed a simple, rapid method for purification of beta-glucuronidase from human liver in order to facilitate the study of its biochemical structure and pathophysiologic roles in both cholelithiasis and carcinogenesis. The procedure includes the following steps: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation, and (4) immunoaffinity chromatography employing murine anti-human beta-glucuronidase monoclonal IgG binding to tresyl-activated agarose. beta-Glucuronidase constitutes 1.3 mg per 100 g of wet liver tissue. The enzyme can be purified with a 10% overall yield and overall purification of 5000-fold in a 2-day cycle on a fairly large scale by the method described. Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by protein blotting.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Solubilization and purification of rat liver 5''-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.     

A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties

Protein G, an IgG-binding molecule, was prepared from the cell walls of a group G streptococcal strain, G-148. The protein could be extracted from the cells by papain digestion and purified by the sequential use of ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Sepharose-coupled human IgG, and gel chromatography on Sephadex G-200. Two protein bands with similar molecular weight, 34,000 and 36,000, were obtained when analyzing the pure protein G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield using this purification scheme was 27% of the protein G solubilized from the cells or 70 micrograms/ml packed bacteria. The Stokes radius and frictional ratio of protein G were determined to 3.53 nm and 1.64, respectively, suggesting an elongated fibrous molecule. The protein did not contain any intrachain disulfide bonds. The amino acid composition of protein G was determined and was found to be different from that of protein A, the well known staphylococcal IgG-binding protein. The equilibrium constants of the reactions between protein G and human, rabbit, mouse, and goat polyclonal IgG, determined by Scatchard plots, ranged between 1 X 10(10) and 7 X 10(10), for rat polyclonal IgG 1.4 X 10(9), and human monoclonal IgG1, IgG2, IgG3, and IgG4 between 2 X 10(9) and 6 X 10(9). These affinity constants were always greater than the corresponding values for protein A. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10.     

Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences

Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.     

Soluble guanylate cyclase from rat lung exists as a heterodimer

The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.     

Brain nicotinic receptors isolated by a monospecific antibody against a synthetic alpha-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody.

Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain. Nicotinic receptors isolated from both immunoaffinity columns showed major bands (silver-stained) at 55K and 70K. Using anti-S3 serum as probe, Western blots of nicotinic receptors isolated by the two immunoaffinity gels also showed major bands at 55 and 70K. However, Western blots of fresh brain extracts revealed a major band at 80K and minor bands at 55K and 70K. These results show similar nicotinic cholinergic receptor proteins isolated by the anti-S3 and 422F11 anti-idiotypic antibodies; 80K was dominant only when fresh brain extract was subjected to Western blotting without prior immunoaffinity purification.     

Purification of human plasma inactive renin by immunoaffinity chromatography on profragment-specific IgG

Inactive renin was purified to apparent homogeneity from human plasma by ion exchange, gel filtration, Affi-Gel blue, immunoaffinity chromatography on profragment-specific IgG coupled to Sepharose, and preparative HPLC. By this method, a 460000-fold purification was obtained. The purified renin was totally inactive and was activated by trypsin.     

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.     

Purification of selenoprotein P from human plasma

Selenoprotein P was partially purified (> 1000-fold) from human plasma in four chromatographic steps using ⁷⁵Se-labeled selenoprotein P secreted by HepG2 cells in culture as a marker. The purified preparation was injected into mice and monoclonal antibodies, which precipitated the labeled protein, were generated. Neither of two different monoclonal antibodies had cross-reactivity with plasma from five animal species. Antibodies were coupled to agarose, and selenoprotein P was purified from human plasma by immunoaffinity chromatography followed by chromatography on heparin agarose. With two different matrix-bound monoclonal antibodies, the purification procedure gave two bands on SDS-PAGE with mobilities corresponding to 61 and 55 kDa. Both bands stained for carbohydrate and showed increased electrophoretic mobility after enzymatic deglycosylation. Immunoaffinity chromatography removed approx. one-third of the selenium from plasma or 0.4 μmol Se/l at a total selenium concentration of 1.1 μmol/l, indicating that selenoprotein P constituted this proportion of total plasma selenium in healthy US blood donors.     

One-step purification of nisin A by immunoaffinity chromatography.

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.     

亲和层析纯化HepG_2细胞分泌的PAI－1

本文报道用抗ＰＡＩ－１单克隆抗体（ＭｃＡｂ）亲和层析建立了纯化ＰＡＩ－１的简便方法。经免疫亲和层析,ＳｅｐｈａｃｒｙｌＳ２００凝胶过滤,从ＨｅｐＧ２细胞培液中分离到糖基化和非糖基化两种形式的ＰＡＩ－１,回收率为８４％,ＰＡＩ－１比活性６．１×１０４ＩＵ／ｍｇ。糖基化ＰＡＩ－１分子量为５０ｋＤ,比活性５．８×１０４ＩＵ／ｍｇ。非糖基化ＰＡＩ－１分子量４３ｋＤ,占总ＰＡＩ－１３０％,仍具有ＰＡＩ－１活性。用ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ亲和层析进一步纯化得到ＳＤＳ－ＰＡＧＥ纯的糖基化ＰＡＩ－１。     

Purification of grapevine flavescence dorée MLO (Mycoplasma-like organism) by immunoaffinity

To date MLO (Mycoplasma-like organism) remain non-culturable organisms and are difficult to extract in good conditions of purity and conservation from infected hosts (plants or leafhopper vectors). An immunoaffinity procedure that permits the purification of large quantities of Grapevine Flavescence dorée MLO (FD-MLO) is described, with covalently bound and oriented IgG molecules of a previously obtained anti-FD-MLO monoclonal antibody and elution of antigens in alkaline conditions. Evidence for purity and integrity of the eluted MLO is presented. The two main antigenic components detected by rabbit polyclonal antibodies to FD-MLO were shown to be different proteins and to contain different epitopes with the use of different monoclonal antibodies. DNA extracted from the purified FD-MLO fraction hybridized with an FD-MLO DNA-specific probe.     

Two-step purification of mouse kidney ornithine decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Purification of an active EGF receptor kinase with monoclonal antireceptor antibodies

A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.     

Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Efficient purification of somatomedin-C/insulin-like growth factor I using immunoaffinity chromatography

Somatomedin-C/insulin-like growth factor I was purified from human plasma using a monoclonal antibody affinity column. Combining immunoaffinity chromatography with standard protein purification methods resulted in an overall recovery of 18%. The 35 micrograms of somatomedin-C/insulin-like growth factor I purified from 500 ml of plasma appeared as a single band when analyzed by polyacrylamide gel electrophoresis and could be used in radioimmunoassay and receptor binding studies.     

The rabbit progesterone receptor. Evidence for a single steroid-binding subunit and characterization of receptor mRNA

Monoclonal antibodies were used to study the structure and the biosynthesis of the rabbit progesterone receptor. Proteins in nonfractionated uterine cytosol were submitted to gel electrophoresis in denaturing conditions, transferred onto nitrocellulose, and reacted with monoclonal antireceptor antibodies and 125I-protein A. A single 110,000-dalton protein was observed when precautions were taken during homogenization of the uteri and protease inhibitors used. Smaller forms of receptor (essentially of 79,000 daltons but also of 72,000 and in some experiments of 64,000 daltons) were present when these precautions were not observed and thus probably arose from artifactual proteolysis of receptor. When poly(A)+ RNA from rabbit uterus was translated in a reticulocyte lysate and the radioactive proteins precipitated by the antireceptor monoclonal antibodies, a radioactive protein of 110,000 daltons was also observed. Further evidence that this protein was the product of the translation of progesterone receptor mRNA was obtained by precipitation and immunoaffinity purification with several antireceptor monoclonal and polyclonal antibodies, inhibition of immunoprecipitation by purified receptor and its absence in a receptor-poor tissue (liver). Estrogen treatment is known to increase the concentration of progesterone receptor. RNA translation experiments showed that this effect is due to an increase in the concentration of receptor mRNA. The size of this messenger RNA was studied by sucrose gradient ultracentrifugation, followed by mRNA translation, and specific immunoprecipitation: progesterone receptor mRNA was found by this method to sediment at 20 S.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

虎血清免疫球蛋白（IgG）的纯化及纯度、活性鉴定

目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。