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Biomedical subjects

P Bailon

Publications and source records attributed to P Bailon.

29 records · Page 2Linked to original sources
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↗
PubMedDate not supplied

Fluidized-bed receptor-affinity chromatography.

A multipurpose fluidized-bed receptor-affinity purification system based upon the biological recognition between an immobilized receptor and its soluble protein ligands is described. The fluidized affinity sorbent consists of a soluble form of interleukin-2 receptor chemically bonded to an aldehyde derivative of controlled pore glass beads, which have a pore diameter of 1000 A and a particle density of 1.2-1.3 g/mL. The fluidized-bed separation device used in this study consists of a specially designed column fitted at the inlet end with a perforated distributor plate covered with a screen and the top outlet with an adjustable piston. The fluidized-bed consisting of a loose gel matrix permits the unimpeded passage of cell debris and particulate matter, while the target protein is captured by the affinity beads. Purification of the humanized-anti-Tac monoclonal antibody is used as a model system to determine the operational parameters. Also, fluidized-bed receptor-affinity chromatography has been successfully employed in the purification of recombinant interleukin-2 and single chain anti-Tac(Fv)-Pseudomonas exotoxin immunotoxin from unclarified inclusion body extracts. Overall, fluidized-bed receptor-affinity chromatography is found to be a productive affinity method suitable for the purification of recombinant human interleukin-2 and related molecules.

ADP Ribose Transferases↗
PubMedDate not supplied

Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C.

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.

Animals↗
PubMedDate not supplied

Affinity purification and characterization of anti-Tac(Fv)-C3-PE38KDEL: A highly potent cytotoxic agent specific to cells bearing IL-2 receptors.

A chimeric, single chain antibody fused immunotoxin, denoted anti-Tac(Fv)-C3-PE38KDEL, was engineered and expressed in Escherichia coli. The microbially expressed anti-Tac(Fv)-C3-PE38KDEL was solubilized from inclusion bodies using guanidine hydrochloride, and subsequently refolded in a redox buffer via thiol/disulfide exchange. The recombinant immunotoxin from the crude extract was purified employing receptor-affinity chromatography, which is based upon biological function and involved the immobilized p55 subunit of human IL-2 receptor. The cytotoxic activity of this immunotoxin was measured by the IL-2 dependent phytohemagglutinin (PHA) blast proliferation inhibition and HUT-102 protein synthesis inhibition assays, in which the IC50 values were 41.5 and 0.8 pM, respectively. The biochemical homogeneity and authenticity of the purified material were determined by gel permeation chromatography, amino acid composition and N-terminal sequence analyses, SDS-PAGE, isoelectric focusing, and Western blotting. The receptor-affinity-purified immunotoxin was shown to be highly effective in specifically killing cells bearing IL-2 receptors. Anti-Tac(Fv)-C3-PE38KDEL is a powerful immunosuppressant which may be a potentially useful therapeutic agent in the prevention of allograft rejection and in the treatment of autoimmune diseases. Another anticipated application of this fusion protein is as a chemotoxin in the treatment of some forms of cancer.

ADP Ribose Transferases↗