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P Bailon

Publications and source records attributed to P Bailon.

16 recordsLinked to original sources
PubMed1992

Membrane-based receptor affinity chromatography.

Membrane-based receptor affinity chromatography (MRAC), which utilizes the molecular recognition between an immobilized receptor and its soluble protein ligand, has been developed for the purification of human interleukin-2 and related biomolecules. The multi-purpose affinity membrane used in this study consisted of a soluble form of interleukin-2 receptor (IL-2R) chemically bonded to hollow-fiber membranes in an oriented fashion. A model system involving anti-Tac-H (a humanized monoclonal antibody to IL-2R) was used to study the important factors influencing the performance of MRAC, including support morphology, mass transfer rate and adsorption kinetics. All three are shown to be highly efficient. MRAC has been successfully applied to the purification of anti-Tac-H, recombinant human interleukin-2 (rIL-2) and interleukin 2-Pseudomonas exotoxin fusion protein (IL2-PE40). Overall, MRAC was found to be a viable, scalable and extremely productive affinity purification method.

Adsorption↗
PubMed1991

Reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac in cynomolgus monkeys.

The anti-Tac mAb has been shown to bind to the p55 chain of the IL-2R, block IL-2 binding and inhibit T cell proliferation. A humanized form of anti-Tac (HAT) has been constructed that retains the binding properties of murine anti-Tac (MAT). These two mAb were evaluated in cynomolgus monkeys to compare relative immunogenicity and pharmacokinetic properties. Monkeys treated with HAT daily for 14 days exhibited anti-HAT antibody titers which were 5- to 10-fold lower than their MAT-treated counterparts and these antibodies developed later than in the MAT-treated monkeys. Two of four monkeys receiving a single injection of MAT developed anti-MAT antibodies, whereas none of four monkeys developed antibodies after a single treatment with HAT. In monkeys injected with either HAT or MAT daily for 14 days, the anti-antibody titers induced were inversely related to the amount of anti-Tac administered. Antibodies that developed against MAT were both anti-isotypic and anti-idiotypic, whereas those developed against HAT appeared to be predominantly anti-idiotypic. The pharmacokinetic properties, that is the half-life and area under the curve values, of HAT were also significantly different from those of MAT. The area under the curve values for HAT in naive monkeys were approximately twofold more than those for MAT, and the mean serum half-life of HAT was 214 h, approximately four- to fivefold more than MAT. These pharmacokinetic values were reduced in monkeys previously sensitized with HAT or MAT suggesting that the presence of anti-antibodies altered these parameters.

Animals↗
PubMed1991

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.

Amino Acid Sequence↗
PubMed1990

Application of receptor-affinity chromatography to bioaffinity purification.

Receptor-affinity chromatography based upon the receptor-ligand interactions has been utilized for the purification of recombinant human interleukin-2 (rIL-2) from microbial and mammalian sources. The receptor-affinity purification process of rIL-2 is used as a model system to demonstrate the utility of this approach for the purification of recombinant proteins. The receptor-affinity purified biomolecule is shown to be biochemically and biologically more homogeneous than the immunoaffinity purified material.

Amino Acids↗
PubMed1990

The use of proteolysis and direct N-terminal sequence analysis to study human interleukin-2/receptor interaction on solid support.

An immobilized interleukin-2 receptor which is capable of binding interleukin-2 and suitable for direct N-terminal sequence analysis was employed to study interleukin-2/receptor interactions. Sensitive tryptic sites on the immobilized receptor and its interleukin-2 complex were identified by sequence analyses and compared. The results have revealed that the N-terminal region of interleukin-2 is not involved in receptor binding and the peptide segment covering residues 36-39 in the receptor is probably near or involved in the interleukin-2 binding site. The rapidity and simplicity make this solid phase sequence approach a good method for analyzing interleukin-2/receptor interaction and may be suitable for studying other protein-ligand interactions.

Amino Acid Sequence↗
PubMed1990

Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins.

Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays. The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2. Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered. Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2. The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.

ADP Ribose Transferases↗
PubMed1988

Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor.

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation.

ADP Ribose Transferases↗
PubMed1988

Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor.

Recombinant technology has facilitated the production of two soluble forms of human interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two IL-2Rs, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug screening assays and the receptor-affinity purification of human recombinant interleukin-2.

Amino Acids↗
PubMed1988

Structural analysis of recombinant soluble human interleukin-2 receptor. Primary structure, assignment of disulfide bonds and core IL-2 binding structure.

A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed.

Amino Acid Sequence↗
PubMed1988

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.

Binding, Competitive↗
PubMed1977

Affinity purification methods. V1. A novel and rapid isolation procedure for lysozymes.

Lysozymes from animal and plant sources have been purified on agarose derivatives containing a phenylacetyl ligand. While this adsorbent can also bind chymotrypsin, the use of imidazole permits separation of lysozymes from the protease. The phenylacetyl-agarose is a general affinity sorbent bearing a ligand of non-biochemical origin in contrast to those, for example, which use nucleotide cofactors. By careful selection of desorption conditions, different enzymes can be purified with the same sorbent. Results also suggest that binding of lysozymes to an affinity ligand does not require a leash structure between ligand and support matrix.

Chromatography, Affinity↗