Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.     

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10⁶ cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.     

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

Abstract

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.     

Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.     

Studies on ram acrosin. Activation of proacrosin accompanying the isolation of acrosin from spermatozoa, and purification of the enzyme by affinity chromatography.

1. A previously described, freeze-dried, partially purified ram acrosin preparation was fractionated on a column of Sepharose linked to the acrosin inhibitor p-(p'-aminophenoxypropoxy)benzamidine. Two acrosin fractions were obtained. 2. beta-Acrosin was homogeneous, quite stable at low pH and very stable when freeze-dried. Its molecular weight is about 38000, and it contains about six sugar residues per molecule, but no sialic acid. psi-Acrosin consisted of at least three unstable forms of acrosin. 3. When the entire purification process, starting from collection of semen, was carried out as rapidly as possible, the yield of beta-acrosin was increased and very little psi-acrosin was obtained. 4. In fresh ram semen the acrosin is present as the intra-acrosomal zymogen, proacrosin. After its extraction from spermatozoa autoproteolytic reactions convert proacrosin into beta-acrosin; psi-acrosin appears to be breakdown products of beta-acrosin. 5. When beta-acrosin was passed through a column of Sepharose linked to the non-inhibitory deamidinated analogue of the inhibitor it behaved as a hydrophobic protein. This is consistent with our view that acrosin (as zymogen) occurs in spermatozoa as a membrane-bound protein. 6. Success in the isolation of pure acrosin in high yield calls for an affinity adsorbent with the appropriate subsidiary hydrophobic properties.     

Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier

A novel affinity separation method in an aqueous two-phase system (ATPS) is suggested, using protein conjugated IgG as a ligand. For verification of the proposed approach, horseradish peroxidase (HRP) and human IgG was used as a ligand carrier and affinity ligand, respectively. The partition of the affinity ligand, human IgG, was controlled by the conjugation of HRP. Two ATPSs, one consisting of potassium phosphate (15%, w/w) and polyethylene glycol (PEG, M.W. 1450, 10%, w/w) and the other of dextran T500 (5%, w/w) and PEG (M.W. 8000, 5%, w/w), were used. The conjugated human IgG-HRP favored a PEG-rich top phase, whereas human IgG, rabbit anti-human IgG and goat anti-mouse IgG preferred a salt or dextran-rich bottom phase. Using the conjugated human IgG-HRP, rabbit anti-human IgG was successfully separated into a PEG-rich top phase from the mixture with goat anti-mouse IgG. The appropriate molar ratio between human IgG-HRP and rabbit anti-human IgG was around 3:1 and 1:1 for the salt and dextran-based ATPS, respectively. The dextran-based ATPS showed a better recovery yield and purity than the salt-based ATPS for the range of test conditions employed in this experiment. The yield and purity of the recovered rabbit anti-human IgG were 90.8 and 87.7%, respectively, in the dextran-based ATPS, while those in the salt-based ATPS were 78.2 and 73.2%.     

A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at 99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was 相似文献   

Antibody against nuclear thyroid hormone receptors

Rat liver nuclear thyroid hormone receptor was purified to 700-1600 pmol T3 binding capacity/mg protein by sequentially using hydroxylapatite column, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column, DEAE-Sephadex A-50 column, and heparin-Sepharose column. Serum from a mouse immunized using this purified receptor preparation caused a shift of [125I]T3-receptor peak on glycerol density gradient sedimentation from 3.4 S to approximately 7 S. [125I]T3-receptor complex was immunoprecipitated using this serum and goat anti-mouse IgG. The serum showed reduced ability to immunoprecipitate the globular T3 binding fragment with Stokes radius of 22 A produced by trypsin digestion, a receptor fragment which has core histone and hormone binding but not DNA binding activity. These data indicate the production of anti-nuclear thyroid hormone receptor antibody which mainly recognized epitopes unrelated to hormone and core histone binding domain.     

New aspects of isolation and purification of thyroxine-binding globulin from human biological fluids

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.     

Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.     

Non-specific effects of passive immunization on implantation in the rabbit.

Passive immunization with goat anti-rabbit uteroglobin antiserum prevents implantation in the rabbit. The dose of antiserum was too low to neutralize all of the uteroglobin present on Day 5 of pregnancy, however, and the effect could not be shown to be specific, because 'control' treatments with goat antiserum to chick avidin or normal goat serum also prevented implantation. Non-specific antisera raised in rabbits had little or no effect on implantation. Partial purification of antibodies from the non-specific goat antisera reversed their effect, while anti-uteroglobin gamma globulin still reduced implantation. Fluorescein-labelled gamma globulin fractions of anti-avidin and anti-uteroglobin both bound to blastocysts, but pure FITC-IgG showed binding only of anti-uteroglobin. Both anti-avidin and anti-uteroglobin IgG prevented implantation. It is concluded that the effect on implantation is not necessarily achieved via a specific antigen.     

DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.     

The use of anti-immunoglobulin for the immunoprecipitation of labeled proteins from tissue homogenates

An improved immunochemical procedure for the quantitative isolation of labeled minor proteins from tissue homogenates is worked out and is applied to the isolation of glucose-6-phosphate dehydrogenase from mouse liver. Goat anti-enzyme serum is used as primary reagent, followed by rabbit anti-goat IgG, and not by carrier enzyme as in currently used methods. The resulting immunoprecipitates are analyzed by acrylamide gel electrophoresis, so that only counts in enzyme bands are registered. An equivalent precipitate formed with serum from nonimmunized goat serves as an efficient control for coprecipitation.     

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.     

中试规模纯化海洋芽孢杆菌源脂肽类化合物

本次研究旨在建立经济可行的海洋芽孢杆菌源脂肽类化合物的中试规模纯化工艺。对包括酸化沉淀、甲醇浸提、溶剂沉淀、盐析、萃取、硅胶柱层析和HZ806大孔树脂吸附工艺在内的可放大的成熟单元工艺进行反复试验,考察脂肽类化合物表面活性对单元工艺的影响。严格遵循以高收率为前提循序渐进逐步减少杂质的原则,组合上述单元工艺对目标产物进行提取和纯化,并最终获得高纯度脂肽样品。新工艺可从1 t海洋芽孢杆菌Bacillus marinus B-9987的发酵液中,以百克量级的规模制备87.51%–100%纯度的脂肽类化合物样品,收率81.73%。本研究首次实现了高纯度的海洋芽孢杆菌源脂肽类化合物的百克量级制备;允许发酵生产阶段使用天然培养基,缓解了脂肽中游发酵生产和下游大规模纯化之间的矛盾;且各单元工艺规避了脂肽类化合物水溶液的乳化起泡和不经济的大体积水溶液蒸发浓缩。新工艺实用可行,经济合理。     

Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91–95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11–13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.     

Solid-phase biotinylation of antibodies

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.     

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.     

A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity.

A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.