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G K Ehrlich

Publications and source records attributed to G K Ehrlich.

8 recordsLinked to original sources
PubMed2001

Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display.

A proof-of-principle study was initiated to determine whether phage-display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography and to identify a peptide or peptidomimetic for use as a Protein A alternative in the affinity purification of monoclonal antibodies. The constant region of humanized anti-Tac (HAT), prepared by pepsin digestion and receptor-affinity chromatography, was used as the target for phage display in this study. As such, 20 phage-derived peptide sequences were identified from four rounds of biopanning with two linear phage-display libraries (7-mer, containing 100 copies of 2 x 10(9) sequences and 12-mer, containing 70 copies of 1.4 x 10(9) sequences). Five peptides were synthesized for use as affinity ligands, based on sequence homology to Protein A, sequence redundancy, and amino acid motifs. The best HAT binding immobilized peptide was EPIHRSTLTALL. The best-fit analysis of this peptide sequence with Protein A yielded an alignment well within the Fc binding domain of Protein A. These results suggest that phage display can serve as a tool in the identification of peptides as model ligands for affinity chromatography.

Amino Acid Sequence↗
PubMed1998

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display.

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.

Amino Acid Sequence↗
PubMed1998

Functional significance of cysteine residues in the delta opioid receptor studied by site-directed mutagenesis.

Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the delta opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned delta opioid receptor using site-directed mutagenesis. The wild-type and mutant receptors were expressed stably in Chinese hamster ovary cells. The two extracellular cysteine residues and the six located in transmembrane domains were mutated to serine or alanine, one at a time. Replacement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti delta opioid receptor antibodies in washed cell monolayers in one of these mutants (Cys-Ser121), and immunoblots, performed on cell extracts, indicate that the receptor was expressed and seems to be associated with the cell membrane. The existence of an essential extracellular disulfide bridge, previously postulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmembrane cysteines was virtually without effect on the ability of the receptor to bind delta agonists and antagonists. Since there is strong evidence that the transmembrane domains are involved in ligand binding, these results suggest that the cysteine residues, even those near or at the binding site, are not essential for receptor binding. Furthermore, these results support the idea that the striking effects of sulfhydryl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydryl groups of cysteine residues by these reagents.

Alanine↗
PubMed1994

Purification and partial amino acid sequence of a 28 kDa cyclophilin-like component of the rat liver sigma receptor.

The sigma (sigma) receptor, a putative non-opioid receptor site which has been suggested to function as a neuromodulator of dopaminergic and NMDA systems, and is found in brain, liver and many other tissues, has been purified > 560-fold from a detergent-solubilized rat liver membrane preparation by affinity chromatography, using an affinity matrix prepared from an oximino derivative of haloperidol. The affinity column selectively retained principal components of M(r) 28 kDa, 40 kDa and 65 kDa that could be eluted from the column with sigma-selective ligands, specifically dextrallorphan and haloperidol. After dialysis and concentration by ultrafiltration, a loss in density of the 65 kDa component and an increase in the 28 kDa and 40 kDa components was observed. A 15 amino acid N-terminal sequence was obtained for the 28 kDa protein which is identical to the N-terminal sequence of the 17 kDa rat cyclophilin A, a cytosolic protein, suggesting that a critical component of the rat liver sigma receptor may be a cyclophilin. These results support the suggestion that sigma receptors are a key link between the central nervous system and the immune system.

Amino Acid Isomerases↗
PubMed1993

N-(1-arylpropionyl)-4-aryltetrahydropyridines, a new class of high-affinity selective sigma receptor ligands.

A series of N-(1-arylpropionyl)-4-aryl-1,2,3,6-tetrahydropyridines, prepared by simple Mannich condensations, have been found by radioligand binding assays to have moderate to high affinity (IC50 0.5-500 nM) for bovine cerebellar sigma receptor/binding sites and no measurable affinity (IC50 > 5000 nM) for bovine striatal D2 receptors. The most active of these compounds rival in potency the most active sigma ligands previously reported. Three of these sigma-active compounds were screened for pharmacological activity under the NIMH-NovaScreen program and showed moderate affinity only for D2 and 5-HT2 receptors among the 40 sites assayed. Since these N-(1-arylpropionyl)-4-aryltetrahydropyridines are structurally related to other potent sigma receptor ligands, in particular haloperidol and 4-phenylpiperidines, these data provide insights into the nature of the essential pharmacophore of the sigma receptor. The selective affinity of these materials for sigma receptors indicates they have potential as prototypes of novel psychotherapeutic medicinal agents, particularly as antipsychotic drugs which would be devoid of debilitating side effects associated with blockade of D2 receptors.

Animals↗
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↗