Affinity purification of natural ligands.

Immobilization of proteins, nucleic acids, and other "bioligands" requires selection of the proper bioligand for the specific application. This decision influences all others: the matrix is chosen and activated by the method that is most appropriate for this specific application and the ligand is coupled under conditions dictated by the activation method and the nature of the ligand (e.g., is it a labile protein or a sturdy enzyme cofactor?). There are many matrices and activation and coupling methods, and new ones are constantly being developed. This unit provides three protocols for activating and coupling proteins and other nucleophilic ligands to beaded agarose gel: cyanogen bromide, p-nitrophenyl chloroformate, and tresyl chloride.

Alginate as a displacer for protein displacement chromatography.

Alginate use in displacement chromatography as a displacer has been studied. The experiments showed that untreated alginate is the basis of potential displacer for displacement chromatography, but needs to be cleaved into smaller chains. Alginate treated with ultrasound, which cleaves alginate into shorter polysaccharide chains, gave better displacement than untreated alginate, while alginate subjected to limited acid hydrolysis gave the best results in displacement chromatography. It was found that the mixture of ovalbumin and beta-lactoglobulin separated well, and several components of ovalbumin were also separated and purified when alginate hydrolysate was used as a displacer. beta-Lactoglobulins A and B, which have the same molecular weight and differ in isoelectric point by only 0.1 pH units, were displaced from Q-Sepharose by alginate hydrolysate.

Studies on oriented and reversible immobilization of glycoprotein using novel boronate affinity gel.

The authors have previously synthesized a novel boronate affinity ligand, catechol [2-(diethylamino)carbonyl-4-bromomethyl]phenylboronate. When this ligand was coupled to cellulose beads, it bound horseradish peroxidase (HRP), a glycoprotein, at pH 7.0. In comparison, commercial m-aminophenylboronic acid-agarose did not bind HRP below pH 8.0. HRP was immobilized in an oriented and reversible fashion using this gel. The immobilized enzyme retained 90.12 per cent of its original activity, probably due to its attachment via the carbohydrate moiety of the enzyme. After repeated use, the activity remaining on the new gel was twice as high as that on conventional m-aminophenylboronic acid-agarose. The column was regenerated easily by washing with dilute acid because of reversibility of the boronate glycol bond.

Dihydrofolate reductase synthesis in the presence of immobilized methotrexate. An approach to a continuous cell-free protein synthesis system.

Dihydrofolate reductase was synthesized in a batch system in the presence of the affinity ligand methotrexate, bound to various matrices. Two types of gel were used: commercial methotrexate-agarose with pores inaccessible for translation machinery and methotrexate-POROS with pores easily accessible for translation reaction mixture components. The transcription/translation reaction was not inhibited by either the immobilized methotrexate or the matrix. The enzyme was synthesized with a high yield and could simultaneously be removed from the reaction mixture by the affinity matrix during the synthesis. With methotrexate-POROS present the reaction probably proceeded mainly in the pores of the gel. Kinetic limitations to the reaction in the presence of the gel were not observed. Active dihydrofolate reductase was eluted from methotrexate-POROS. The activity recovered was higher than dihydrofolate reductase activity synthesized in free solution system. The influence of the presence of immobilized methotrexate on dihydrofolate reductase synthesis will be further studied in a novel type of a continuous protein synthesis system.

Oriented immobilization of restriction endonuclease EcoRI.

Two activated matrices have been developed to determine whether immobilization chemistry can be used to orient proteins on a support. Restriction endonuclease EcoRI from Escherichia coli RY13 (E.C.3.1.23.13) was used as a model in these studies. Thiol-activated Sephadex G-10 was used to couple the EcoRI endonuclease through its free sulfhydryl, while amino-activated Sephadex G-10 was used to couple it randomly through its free carboxyl groups. To determine whether the enzyme was immobilized randomly or specifically, both lower and higher molecular weight substrates were used. The polymerase chain reaction amplified multiplied cloning site region of pBluescript KS obtained using T3 and T7 primers was considered as the small substrate. The plasmid SP64 containing firefly luciferase gene was the large substrate. Immobilized EcoRI preparations were characterized with respect to repeated usage and storage stability. The EcoRI immobilized on thiopropyl-Sepharose 4B could be stored for over 14 days at 4 degrees C without observable loss of activity. In an independent experiment the same gel was used thrice repeatedly without any discernible loss of activity.

Purification of monospecific polyclonal antibodies from hyperimmune bovine whey using immunoaffinity chromatography.

Whey, a by-product of cheese production, is a potential source of proteins. Immunization of dairy cows in mid-lactation with mouse IgG and dinitrophenyl-keyhole limpet hemocyanin resulted in the formation of antibodies to these antigens in both blood serum and milk. The antibodies remained in whey during cheese making, and were isolated by immunoaffinity chromatography on matrices with immobilized antigens. The isolated monospecific antibodies were pure and retained their reactivity to antigens.

Study of cell-free protein synthesis in aqueous two-phase systems.

Protein synthesis in an aqueous two-phase system is reported here as a novel type of extractive bioconversion. Translation of BMV RNA was studied using either rabbit reticulocyte lysate or wheat-germ extract in aqueous dextran-PEG systems. Both polymers inhibited protein synthesis and the translation system partitioned into both phases. In the rabbit reticulocyte two-phase system, protein synthesis reached 30% of that in free solution, while in wheat-germ extract it was 60% of that in free solution. Protein was found mainly in the dextran (lower) phase but its partitioning was related to the polymer concentration, and molecular weight, as well as the ionic strength of the system. Protein synthesis was highly influenced by PEG concentration, potassium chloride concentration, and dextran molecular weight.

Chromatographic purification of immunoglobulin G from bovine milk whey.

We used thiophilic chromatography on T-gel, a resin of the structure agarose-O-CH2CH2SO2CH2 CH2SCH2CH2OH, to purify immunoglobulin G from "sweet" cheese whey. The purity of immunoglobulin G, as indicated by radial immunodiffusion, was 74% after a single chromatography on T-gel. Preparation of samples for adsorption onto thiophilic gels requires only the addition of salt (sodium/potassium sulfate) to the samples. Thus, this method may be suitable for large-scale whey IgG isolation.

Reversible immobilization of antibodies on magnetic beads.

A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.

Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.

Immobilization of perfluoroalkylated enzymes in a biologically active state onto Perflex support.

Perflex has been introduced by E. I. du Pont de Nemours and Co., Inc., as a new fluorocarbon-based technology for protein immobilization. Due to the hydrophobic character of the support, however, significant loss of enzymatic activity may occur upon immobilization of certain enzymes, which appears to be due to a large conformational change of the protein ("inversion"). Pretreatment of the Perflex support with a neutral fluorosurfactant lessened the surface hydrophobicity, thus decreasing the hydrophobic interaction between the support and the protein. Modification of enzymes with a high number of fluorocarbon residues, which forms a hydrophobic "envelope" around the protein, also appears to prevent enzyme inactivation upon immobilization on Perflex support. Moreover, preactivation of the support with either perfluorooctylpropylisocyanate or reactive poly(fluoroalkyl) sugar reagents greatly improves the enzyme particle activity by increasing the amount of immobilized enzyme. Fluorosurfactant treatment of the support activated with perfluorooctylpropylisocyanate improves the retention of activity for sensitive enzymes such as alpha-chymotrypsin and increases the wetability and ease of handling of the Perflex particles.

Affinity chromatography for protein isolation.

Thousands of reports concerning protein purification have appeared in the past year, and over 150 of these involved, at least in part, the affinity chromatography process. Immobilized membrane affinity chromatography, temperature-programmed elution, and centrifugal affinity chromatography are among the most significant new techniques amid the myriads of applications in this mature field.

A calcium-selective optrode based on the fluorescence of dansylated troponin.

Troponin C (TnC) is covalently labeled with a fluorescent probe molecule dansylaziridine and immobilized on polyethyleneimine-impregnated cellulose. An optrode is constructed by attaching the treated cellulose membrane to the distal end of a bifurcated fiber-optic cable. Because fluorescence is enhanced as Ca+2 binds with the immobilized dansylated troponin (TnCDANZ), this system can be used as a calcium sensor for monitoring plasma Ca+2 levels.

S-Zephyr, a new high performance ion exchange chromatography column matrix.

S-Zephyr, a new column material for high performance cation exchange chromatography of proteins, is compared with Mono-S. The comparison is based on a retentivity study, a model separation of an artificial protein mixture, a sample load capacity experiment and the development of the separation performance at a column overload situation. S-Zephyr is found to be a good matrix for cation exchange chromatography, for analytical separations as well as for small and large scale protein purification applications.

Centrifugal affinity chromatography.

A new technique termed centrifugal affinity chromatography (CAC) is presented in this paper. CAC combines a high flow-rate, created by centrifugal force, with the specificity of affinity chromatography. This technique has been used for the purification of human immunoglobulin G. Furthermore this technique has been used to remove human albumin from serum and the effect of centrifugal force, ionic strength and pH has been studied. A test for determining the percentage of glycosylated hemoglobin in hemolysates has also been developed. This test, employing centrifugal chromatography, is more than three times faster than commonly used gravity flow methods.

Dye-ligand centrifugal affinity chromatography.

A fast method for the screening of a large number of immobilized dyes for the purification or binding of proteins called dye-ligand centrifugal affinity chromatography, is described. The ease and speed of this method is demonstrated by screening 65 immobilized dyes for the binding of purified goat IgG. Two immobilized dyes (Drimarene Blue K-R and Drimarene Rubine R/K-5BL) with a high affinity for goat IgG were found to bind specifically the Fc-fragment of the IgG.

Centrifugal column chromatography for small sample purification.

We evaluated several gel filtration materials for suitability in spun column chromatography a rapid desalting method. The materials were evaluated for resistance to column cracking and to radial shrinkage for sample recovery and for salt separation. A series of graphs can be derived from these evaluations which relates the gel heights to sample volumes for maximum recovery and salt separation while maintaining low sample dilution.