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Richard A Houghten - One of the best experts on this subject based on the ideXlab platform.
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the use of soluble Synthetic Peptide combinatorial libraries to determine antigen recognition of t cells
Journal of Peptide Research, 2009Co-Authors: Bernhard Hemmer, J. R. Appel, Richard A Houghten, Clemencia Pinilla, J Pascal, Roland MartinAbstract:: T cells identify by their T-cell receptor (TCR) short Peptides in the context of major histocompatiblity complex (MHC) molecules. The interaction of the trimolecular complex composed of the TCR and MHC bound Peptide was extensively studied using substitution analogs of the original Peptide ligands to define those residues important for T-cell recognition in the Peptide chain. This approach has led to the observation that T-cell recognition is highly flexible and that many different Peptides can be recognized by an individual TCR. Others and we have recently introduced Synthetic Peptide combinatorial libraries (SCL) to investigate T-cell recognition. Here we review the SCL-based approaches and describe our current techniques for mapping TCR motifs for CD4+ T cells. The implications of our findings for the understanding of T-cell recognition, as well as for future applications to study T-cell responses in infectious diseases, autoimmune disorders and cancer are discussed.
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acetalins opioid receptor antagonists determined through the use of Synthetic Peptide combinatorial libraries
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Colette T. Dooley, N N Chung, Peter W Schiller, Richard A HoughtenAbstract:Abstract A Synthetic Peptide combinatorial library made up of 52,128,400 hexaPeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual Peptides with mu receptor affinity were found using an iterative process for successively determining the most active Peptide mixtures. Upon completion of this iterative process, the three Peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These Peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These Peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).
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identification of substrate analog trypsin inhibitors through the screening of Synthetic Peptide combinatorial libraries
Biochemistry, 1993Co-Authors: Jutta Eichler, Richard A HoughtenAbstract:Synthetic Peptide combinatorial libraries (SPCLs), which are made up in total of tens to hundreds of millions of Peptides, enable the systematic screening for biologically active Peptides in virtually all in vitro and even in vivo assay systems. In the current study, the applicability of this method to the identification of Peptide enzyme inhibitors was investigated using trypsin as the model enzyme. A specifically designed library of hexaPeptide mixtures was synthesized on cotton carriers and screened. The Synthetic approach, using cotton as a solid support, was modified so that the deprotected Peptides remained attached to the cotton carrier until they were released into solution directly prior to being assayed. Following an iterative process of synthesis and screening, in which all of the positions of the sequence were successively defined, a number of individual hexaPeptides with trypsin inhibitory activity were identified. The most active, defined individual Peptide sequence was then reincorporated into a new library, now made up of dodecaPeptide mixtures. The iterative screening and synthesis of this library led to a dodecaPeptide with improved inhibitory activity when compared to the hexaPeptide from which it was derived.
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Synthetic Peptide combinatorial libraries spcls identification of the antigenic determinant of β endorphin recognized by monoclonal antibody 3e7
Gene, 1993Co-Authors: Clemencia Pinilla, J. R. Appel, Richard A HoughtenAbstract:Abstract The use of Synthetic Peptide combinatorial libraries (SPCLs), each composed of tens of millions of Peptides, is described here for the identification of bioactive Peptides. The identification of optimal Peptide sequences is achieved through the screening of SPCLs in solution, each element of which is composed of more than 105 nonsupport-bound Peptides in approximately equimolar representation, along with an iterative synthesis and screening process. Using an SPCL composed in total of 52 128 400 nonacetylated hexaPeptides, along with an iterative selection process based on competitive ELISA, we identified the antigenic determinant of β-endorphin recognized by monoclonal antibody (mAb) 3E7. These results will be compared with the results found by others investigating mAb 3E7 using different Peptide library approaches.
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the use of positional scanning Synthetic Peptide combinatorial libraries for the rapid determination of opioid receptor ligands
Life Sciences, 1993Co-Authors: Colette T. Dooley, Richard A HoughtenAbstract:Abstract The application of a new Synthetic Peptide combinatorial library (SPCL) is described. This library, termed a positional scanning SPCL, contains six positional SPCLs, each of which contains all possible hexameric combinations of 18 of the 20 natural L-amino acids (18 6 = 34, 012, 224 Peptides). Each positional SPCL (O 1 XXXXX-NH 2 , XO 2 XXXX-NH 2 , XXO 3 XXX-NH 2 , XXXO 4 XX-NH 2 , XXXXO 5 X-NH 2 , and XXXXXO 6 -NH 2 ) was used to determine the most active amino acid for the six positions of a hexamer. Combinations of these amino acids were used to synthesize 24 individual Peptides, which were then tested for activity. The most active Peptide found corresponded to a hexameric analogue of methionine-enkephalin. Results obtained in this study are compared to those obtained using the SPCL described earlier (1) (O 1 O 2 XXXX-NH 2 ), and the subsequent iterative process.
Xiang-qin Liu - One of the best experts on this subject based on the ideXlab platform.
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protein c terminal labeling and biotinylation using Synthetic Peptide and split intein
PLOS ONE, 2009Co-Authors: Gerrit Volkmann, Xiang-qin LiuAbstract:Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small Synthetic Peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein Peptide containing the desired chemical groups.
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novel split intein for trans splicing Synthetic Peptide onto c terminus of protein
Journal of Biological Chemistry, 2009Co-Authors: Julia H Appleby, Gerrit Volkmann, Kaisong Zhou, Xiang-qin LiuAbstract:Conventional split inteins have been useful for trans-splicing between recombinant proteins, and an artificial S1 split intein is useful for adding Synthetic Peptide onto the N terminus of recombinant proteins. Here we have engineered a novel S11 split intein for trans-splicing Synthetic Peptide onto the C terminus of recombinant proteins. The C-intein of the S11 split intein is extremely small (6 amino acids (aa)); thus it can easily be produced together with a Synthetic C-extein to be added to the C terminus of target proteins. The S11 intein was derived from the Ssp GyrB intein after deleting the homing endonuclease domain and splitting the remaining intein sequence near the C terminus, producing a 150-aa N-intein (IN) and a 6-aa C-intein (IC). Its trans-splicing activity was demonstrated first in Escherichia coli cells and then in vitro for trans-splicing between a Synthetic Peptide and a recombinant protein. The in vitro trans-splicing reaction exhibited a typical rate constant of (6.9+/-2.2)x10(-5) s(-1) and reached a high efficiency of approximately 80%. This S11 split intein can be useful for adding any desirable chemical groups to the C terminus of a protein of interest, which may include modified and unnatural amino acids, biotin and fluorescent labels, and even drug molecules.
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novel split intein for trans splicing Synthetic Peptide onto c terminus of protein
Journal of Biological Chemistry, 2009Co-Authors: Julia H Appleby, Gerrit Volkmann, Kaisong Zhou, Xiang-qin LiuAbstract:Conventional split inteins have been useful for trans-splicing between recombinant proteins, and an artificial S1 split intein is useful for adding Synthetic Peptide onto the N terminus of recombinant proteins. Here we have engineered a novel S11 split intein for trans-splicing Synthetic Peptide onto the C terminus of recombinant proteins. The C-intein of the S11 split intein is extremely small (6 amino acids (aa)); thus it can easily be produced together with a Synthetic C-extein to be added to the C terminus of target proteins. The S11 intein was derived from the Ssp GyrB intein after deleting the homing endonuclease domain and splitting the remaining intein sequence near the C terminus, producing a 150-aa N-intein (IN) and a 6-aa C-intein (IC). Its trans-splicing activity was demonstrated first in Escherichia coli cells and then in vitro for trans-splicing between a Synthetic Peptide and a recombinant protein. The in vitro trans-splicing reaction exhibited a typical rate constant of (6.9 ± 2.2) × 10–5 s–1 and reached a high efficiency of ∼80%. This S11 split intein can be useful for adding any desirable chemical groups to the C terminus of a protein of interest, which may include modified and unnatural amino acids, biotin and fluorescent labels, and even drug molecules.
Clemencia Pinilla - One of the best experts on this subject based on the ideXlab platform.
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the use of soluble Synthetic Peptide combinatorial libraries to determine antigen recognition of t cells
Journal of Peptide Research, 2009Co-Authors: Bernhard Hemmer, J. R. Appel, Richard A Houghten, Clemencia Pinilla, J Pascal, Roland MartinAbstract:: T cells identify by their T-cell receptor (TCR) short Peptides in the context of major histocompatiblity complex (MHC) molecules. The interaction of the trimolecular complex composed of the TCR and MHC bound Peptide was extensively studied using substitution analogs of the original Peptide ligands to define those residues important for T-cell recognition in the Peptide chain. This approach has led to the observation that T-cell recognition is highly flexible and that many different Peptides can be recognized by an individual TCR. Others and we have recently introduced Synthetic Peptide combinatorial libraries (SCL) to investigate T-cell recognition. Here we review the SCL-based approaches and describe our current techniques for mapping TCR motifs for CD4+ T cells. The implications of our findings for the understanding of T-cell recognition, as well as for future applications to study T-cell responses in infectious diseases, autoimmune disorders and cancer are discussed.
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Synthetic Peptide combinatorial libraries spcls identification of the antigenic determinant of β endorphin recognized by monoclonal antibody 3e7
Gene, 1993Co-Authors: Clemencia Pinilla, J. R. Appel, Richard A HoughtenAbstract:Abstract The use of Synthetic Peptide combinatorial libraries (SPCLs), each composed of tens of millions of Peptides, is described here for the identification of bioactive Peptides. The identification of optimal Peptide sequences is achieved through the screening of SPCLs in solution, each element of which is composed of more than 105 nonsupport-bound Peptides in approximately equimolar representation, along with an iterative synthesis and screening process. Using an SPCL composed in total of 52 128 400 nonacetylated hexaPeptides, along with an iterative selection process based on competitive ELISA, we identified the antigenic determinant of β-endorphin recognized by monoclonal antibody (mAb) 3E7. These results will be compared with the results found by others investigating mAb 3E7 using different Peptide library approaches.
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rapid identification of high affinity Peptide ligands using positional scanning Synthetic Peptide combinatorial libraries
BioTechniques, 1992Co-Authors: Clemencia Pinilla, J. R. Appel, P Blanc, Richard A HoughtenAbstract:We describe here a conceptually unique set of individual Synthetic Peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL), that can be used for the rapid (i.e., a single day) identification of Peptide sequences that bind with high affinity to antibodies, receptors or other acceptor molecules. The PS-SPCL described here is made up of six individual positional Peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixtures. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.
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the use of Synthetic Peptide combinatorial libraries for the identification of bioactive Peptides
BioTechniques, 1992Co-Authors: Richard A Houghten, Sylvie E. Blondelle, Julio H. Cuervo, Colette T. Dooley, J. R. Appel, Clemencia PinillaAbstract:The systematic preparation of Synthetic Peptide combinatorial libraries (SPCLs), each composed of tens of millions of Peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal Peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound Peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-Peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a Peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial Peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity Peptide ligands using an opiate radio-receptor binding assay.
Patricia Grasso - One of the best experts on this subject based on the ideXlab platform.
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oral delivery of d leu 4 ob3 and ma d leu 4 ob3 Synthetic Peptide leptin mimetics immunofluorescent localization in the mouse hypothalamus
Brain Research, 2017Co-Authors: Brian M Anderson, Lauren Jacobson, Zachary M Novakovic, Patricia GrassoAbstract:This study describes the localization of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, Synthetic Peptide leptin mimetics, in the hypothalamus of Swiss Webster and C57BL/6J wild-type mice, leptin-deficient ob/ob mice, and leptin-resistant diet-induced obese (DIO) mice. The mice were given [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in 0.3% dodecyl maltoside by oral gavage. Once peak serum concentrations were reached, the mice received a lethal dose of pentobarbital and were subjected to intracardiac perfusion fixation. The brains were excised, post-fixed in paraformaldehyde, and cryo-protected in sucrose. Free-floating frozen coronal sections were cut at 25-µm and processed for imaging by immunofluorescence microscopy. In all four strains of mice, dense staining was concentrated in the area of the median eminence, at the base and/or along the inner wall of the third ventricle, and in the brain parenchyma at the level of the arcuate nucleus. These results indicate that [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 cross the blood-brain barrier and concentrate in an area of the hypothalamus known to regulate energy balance and glucose homeostasis. Most noteworthy is the localization of [D-Leu-4]-OB3 immunoreactivity within the hypothalamus of DIO mice via a conduit that is closed to leptin in this rodent model, and in most cases of human obesity. Together with our previous studies describing the effects of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on energy balance, glucose regulation, and signal transduction pathway activation, these findings are consistent with a central mechanism of action for these Synthetic Peptide leptin mimetics, and suggest their potential usefulness in the management of leptin-resistant obesity and type 2 diabetes in humans.
Colette T. Dooley - One of the best experts on this subject based on the ideXlab platform.
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acetalins opioid receptor antagonists determined through the use of Synthetic Peptide combinatorial libraries
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Colette T. Dooley, N N Chung, Peter W Schiller, Richard A HoughtenAbstract:Abstract A Synthetic Peptide combinatorial library made up of 52,128,400 hexaPeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual Peptides with mu receptor affinity were found using an iterative process for successively determining the most active Peptide mixtures. Upon completion of this iterative process, the three Peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These Peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These Peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).
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the use of positional scanning Synthetic Peptide combinatorial libraries for the rapid determination of opioid receptor ligands
Life Sciences, 1993Co-Authors: Colette T. Dooley, Richard A HoughtenAbstract:Abstract The application of a new Synthetic Peptide combinatorial library (SPCL) is described. This library, termed a positional scanning SPCL, contains six positional SPCLs, each of which contains all possible hexameric combinations of 18 of the 20 natural L-amino acids (18 6 = 34, 012, 224 Peptides). Each positional SPCL (O 1 XXXXX-NH 2 , XO 2 XXXX-NH 2 , XXO 3 XXX-NH 2 , XXXO 4 XX-NH 2 , XXXXO 5 X-NH 2 , and XXXXXO 6 -NH 2 ) was used to determine the most active amino acid for the six positions of a hexamer. Combinations of these amino acids were used to synthesize 24 individual Peptides, which were then tested for activity. The most active Peptide found corresponded to a hexameric analogue of methionine-enkephalin. Results obtained in this study are compared to those obtained using the SPCL described earlier (1) (O 1 O 2 XXXX-NH 2 ), and the subsequent iterative process.
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the use of Synthetic Peptide combinatorial libraries for the identification of bioactive Peptides
BioTechniques, 1992Co-Authors: Richard A Houghten, Sylvie E. Blondelle, Julio H. Cuervo, Colette T. Dooley, J. R. Appel, Clemencia PinillaAbstract:The systematic preparation of Synthetic Peptide combinatorial libraries (SPCLs), each composed of tens of millions of Peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal Peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound Peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-Peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a Peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial Peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity Peptide ligands using an opiate radio-receptor binding assay.
