Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

The 19th biennial meeting of the International Society for Molecular Recognition (ISMR), Affinity 2011, was hosted by Professor Guilherme N. M. Ferreira (IBB/UALG) in Tavira, Portugal from June 16th to 19th, 2011. There were 110 registered participants, including 44 students, from 25 countries, in attendance. As stated by the organizers, the main theme of Affinity 2011 centered on “highlights of the scientific advances in molecular binding and recognition through life sciences, bioengineering and nanotechnologies,” with particular focus on aspects of molecular biorecognition related to cell signaling and differentiation, development and application of devices and affinity-based technologies. Despite an economic climate, similar to that experienced at Affinity 2009 in Iceland, Affinity 2011 was received with great success, thanks to the unwavering commitment of Professor Ferreira and his local organizing committee [co-chair Raquel Aires-Barros (IBB/IST), Ana Azevedo (IBB/IST), Cecilia Roque (Requimte/UNL), Joao Goncalves (IMM/FFUL) and DECHEMA (secretariat)]. In the tradition of past Affinity meetings, Affinity 2011 was host to a welcoming reception, the ISMR/Pierce Affinity Award, the Younger Investigator's Award and a gala dinner featuring traditional Portuguese music, Fado, at the Tavira castle. In addition, two “Travel Awards” were kindly sponsored by the Journal of Molecular Recognition (JMR). The scientific program at Affinity 2011 was packed with 41 speakers in nine sessions covering the following topics over 3 days: (1) kinetics and thermodynamics of biomolecular interactions, (2) evolutionary engineering and combinatorial design for affinity and drug discovery, (3) affinity interactions in cell biology – signaling pathways and networks, (4) affinity and protein–protein interactions in health and disease, (5) computational modeling and biomimetic design and materials, (6) affinity-based bioprocessing, (7) nanotechnology, nanomaterials, micro and nanosystems, (8) single-molecule detection: devices/sensors and in vivo tracking and (9) self-assembly and mechanisms of protein machines. Set in Algarve, the southern region of Portugal known for “sunshine breaks and relaxing holidays,” Klaus Mosbach opened Affinity 2011 with a retrospective on “The history of ISMR”. Alois Jungbauer from the Department of Biotechnology at the University of Natural Resources and Life Science in Vienna received the 2011 ISMR/Pierce Affinity Award for his outstanding contributions to the field of “downstream processing.” His lecture entitled “Staphylococcal protein A and camelid antibody affinity chromatography: engineering principles and surface characterization” described the introduction of specific camelid antibodies as an alternative to protein A-based immunoaffinity purification. Two Travel Awards, kindly donated by the Journal of Molecular Recognition, were given to the student, Jennifer D. Knoop from the University of Houston, TX, and the Post-Doc Graziella El Khoury from the University of Cambridge, UK. The Younger Investigators Award sponsored by Hoffmann-La Roche was organized by A. Cecilia Roque and George Ehrlich. Awards were given to nine younger investigators in recognition of their outstanding presentations and active participation at Affinity 2011: Nima Matias Jokilaakso and Johan Nilvebrant (Sweden), Dorota Smolarek (Poland), Matthias Meininger (Germany), Takafumi Honjo (Japan), Alessandro Cumbo (Switzerland) and Luís de Matos Borlido, João Rodrigo Cardoso Trabuco and Telma Barroso (Portugal). The meeting concluded on June 19th with an invitation to the 20th biennial meeting of the International Society for Molecular Recognition in Vienna, Austria, by Affinity 2013 host and organizer, Alois Jungbauer. The 12 peer-reviewed articles published in this special issue span the spectrum of topics presented at Affinity 2011 and cover many profound aspects of affinity-based science and technology. The first section of this volume is devoted to affinity-based technologies and opens with a review article by Maria Raquel Aires-Barrosa's group summarizing strategies for lectin purification. The subsequent article, written by Ranjini and Vijayalakshmi, describes the adsorption of catalase on two mixed mode ligands and discusses the mechanism involved. The next paper by Tekiner et al. describes the use of cryogels for metal-based affinity chromatography for urease purification from jack beans. A similar cryogel was used by Andaç et al., who prepared a new composite protein-imprinted macroporous cryogel for depletion of albumin from human serum prior to use in proteomic applications. Following is a paper by Czarnecka et al., describes the Engineering of Candida albicans glucosamine-6-phosphate synthase by insertion of His6 sequences, a commonly used affinity tag, for efficient enzyme purification. This section concludes with a paper by Sandoval et al., who discuss the use of general rate model to describe elution relationships in affinity chromatography. Two articles by the group of Maria H. L. Ribeiro are related to enzyme immobilization. The first article by Nunesa et al. is entitled “High-affinity water soluble system for efficient naringinase immobilization in polyvinyl alcohol–dimethyl sulfoxide lens-shaped particles.” The, second, by Furtado et al., is entitled “Hesperidinase encapsulation towards hesperitin production targeting improved bioavailability.” The third section deals with the topic of protein–protein interactions. The first article by Kysilka and Vondrašek discusses the analysis of protein–protein interactions in dimeric structures at the molecular level utilizing chemical composition, binding preferences and residue interaction energies, using AMBER empirical force field, in an attempt to reach a better understanding of these interactions in nature. The high-affinity cohesin–dockerin interactions are the topic of the next paper by Slutzki et al., who developed an indirect ELISA-based approach for comparative measurement of these interactions. The dimerization-dependent autophosphorylation, ligand binding and curcumin inhibition of a liposome reconstituted epidermal growth factor receptor are discussed by Doumiati et al. in the next article. The volume concludes with another article from Maria H. L. Ribeiroa's group entitled “Design of selective production of sophorolipids by Rhodotorula bogoriensis through nutritional requirements” by Ribeiroa et al. We wish to thank all the contributors to this volume. We would also like to thank the reviewers who spared either time or effort for shaping and polishing the manuscripts presented in the following pages. Thanks are also due to the editors of the Journal of Molecular Recognition, the Editor-in-Chief, Prof. Marc H. V. Van Regenmortel, and the Executive Commissioning Editor, Dr. Martin Rothlisberger, for the continuing support of the special relationship between the Journal and ISMR and for publishing these proceedings. Special thanks are due to the JMR staff, in particular, Ms Shiela Flores, Ms Faith Pidduck and Ms Rebecca Ralf, who helped us in overcoming difficulties encountered in the editorial work and achieving the final form of this volume. Special thanks are due to Ed Bayer and Meir Wilchek for reviewing, polishing and bettering this article. The ISMR and meeting organizers are grateful to the University of Algarve, the Portuguese Foundation for Science and Technology (FCT), Hoffmann-La Roche and the Journal of Molecular Recognition for their support of Affinity 2011. Thanks are also due to DECHEMA for organizing the Meeting. We look forward to seeing you at Affinity 2013 in Vienna!

Anti-PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti-PEG IgM was observed using the 30 kDa PEG-derivatized NuGel (single passage). Purified anti-PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti-PEG IgM, in solution and adsorbed on 20 kDa PEG-derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS-PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution-digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N-terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti-PEG IgM fragments were characterized for binding to pegylated interferon alfa-2a by ELISA. The results from these studies suggest that affinity purified anti-PEG IgM and fragments can be used as probes in detection assays PEG functionalized biotherapeutics in pre-clinical and clinical studies. Copyright (c) 2008 John Wiley & Sons, Ltd.

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.

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.

Protein A has long been the ligand of choice in the affinity purification of immunoglobulin G1 (IgG1) monoclonal antibodies (see Notes 1 and 2). However, current research efforts (1–9) have been focused on the discovery of small molecules (peptides or peptidomimetics) that share similar binding characteristics with protein A but are more cost effective owing to small size (for ease of synthesis) and stability (for ease of regeneration). The following methods were developed as part of a proof of principle study (1) to determine whether phage display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography (see Note 3) and (2) to identify a peptide or peptidomimetic for use as a protein A alternative in the affinity purification of monoclonal antibodies. In this study, the constant region (pFc’ fragments; see Note 4) of an IgG1 monoclonal antibody, denoted humanized anti-Tac (HAT), was used as the target for phage display in this study. HAT is a humanized monoclonal antibody against the low-affinity p55 subunit of the interleukin-2 (IL-2) receptor.

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. Copyright © 1998 John Wiley & Sons, Ltd.

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.

The use of photogenerated species to study the interactions between ligands and biological macromolecules, particularly proteins and nucleic acids, originated with the pioneering studies of Westheimer (Singh et al., 1962; Shafer et al., 1966; Westheimer, 1978), and has now become a widely used technique for obtaining insight into the site and nature of such interactions. The idea is to use a suitably modified ligand, or sometimes the ligand itself, to identify the particular macromolecule responsible for the biological activity of the ligand, and hopefully the specific site on the macromolecule where the biologically significant interaction takes place. There are many advantages to a photoaffinity reagent, i.e. a reagent that binds irreversibly to the active site on the relevant macromolecule only on exposure to UV or visible light of the appropriate wavelength, vis a vis a reagent that labels its binding site in the dark through thermal activation. These have been clearly enumerated in Bayley’s excellent monograph (1983), which is required reading for everyone interested in this subject. First, a photoaffinity reagent is generally inert with respect to covalent linkage to its binding site in the dark, allowing assays of biological activity and determination of binding affinity without inducing covalent interaction, which is not the case with reagents for chemical affinity labeling. Second, covalent attachment of the photolabel occurs only on exposure of the ligand t o light, so that the labeling agent can be placed directly at the desired target site prior to activation, minimizing attachment to biologically irrelevant macromolecules present in the system. Thus, the reaction of the photolabel can be directly targeted to its binding site by proper manipulation of the system. Finally, the intermediates generated from the photoaffinity label are in general much more reactive than thermally generated intermediates, and can attack functional groups (particularly C-H bonds) that are normally inert to chemical affinity labeling. In some cases, these photointermediates can be directly identified by standard techniques such as laser flash photolysis, but in most applications of this technique the identity of the specific reactive intermediate has not been directly established experimentally. There are a number of potential pitfalls involved in application of this technique which have been clearly identified (Chowdhry and Westheimer, 1979; Knowles, 1972; Bayley and Knowles, 1977; Schuster et al., 1985). Thus, if a chemically modified ligand is utilized, which is the most common approach, it must be established (usually by competition studies) that the modified ligand interacts at the same biologically relevant site as the unmodified ligand. It must also be demonstrated that the binding or receptor site is not destroyed or deactivated by the light used to activate the photolabel, that the photolabel is chemically inert in the dark with respect to covalent attachment to the binding site, and that non-specific labeling (pseudophotoaffinity labeling) of the macromolecule remote from the binding site is minimal. The latter can be a serious problem when a large concentration of the photolabel is utilized, when the rate of dissociation of the ligand from the binding site is fast, and when the photogenerated reactive intermediate is relatively long-lived. Techniques for assessing the importance of these problems in a given system are discussed by Bayley (1983). There has not been a review on PhotoafJinity Labeling in this journal in recent years. Accordingly, we have surveyed the literature in this area from 1985 to late 1988, and hope that we have located most of the important contributions published during this four-year period. Since the literature on photolabeling of biological systems is vast, we necessarily have been somewhat selective, and cannot claim that we have achieved total coverage of this literature. Following an initial survey of advances in photolabeling methodology and relevant mechanistic studies, we summarize applications of this technique in investigations of specific classes of biologically active systems, e.g., neuronal and hormonal receptors, nucleotides, enzymes, transport proteins, etc.