An engineered IN-1 Fab fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity

The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/κ antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 Fab fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 Fab fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 μM. The affinity of the unmutated IN-1 Fab fragment was 8-fold lower. The engineered Fab fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered Fab fragment as a reagent for promoting axonal regeneration in viv

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Biochimica et Biophysica Acta - Biomembranes 1788 (2009): 1255-1262, doi:10.1016/j.bbamem.2008.12.016.Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na+/Ca2+ exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far- UV circular dichroism and infrared spectra suggest a majority of β-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten β-sheets and two short α- helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.The work was supported by Grants from the US National Science Foundation [MCB 0444598], Fondo Nacional para Investigaciones Científicas y Tecnológicas [PICT-05- 12397 and PICT-05-38073], Consejo Nacional de Investigfaciones Científicas y Técnicas [PIP 5118 and PIP 5593] Secretaría de Ciencia y Técnica Universidad Nacional de Córdoba, Argentina, Fondo Nacional para Ciencia y Técnica [S1-9900009046 and G- 2001000637] and Fundación Polar, Venezuela and The Rhode Island Idea Network of Biomedical Research Excellence (INBRE)

PuLSE:Quality control and quantification of peptide sequences explored by phage display libraries

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use

Anchored Design of Protein-Protein Interfaces

Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders.Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space.This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor

High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells

Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (VH) and light (VL) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of VH and VL domains, further validating the here-reported expression system

NMR Structure of Lipoprotein YxeF from Bacillus subtilis Reveals a Calycin Fold and Distant Homology with the Lipocalin Blc from Escherichia coli

The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic β-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the α-helix that packs in all lipocalins with known structure against the β-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named ‘slim lipocalins’, with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses

Adaptive Response of a Gene Network to Environmental Changes by Fitness-Induced Attractor Selection

Cells switch between various stable genetic programs (attractors) to accommodate environmental conditions. Signal transduction machineries efficiently convey environmental changes to the gene regulation apparatus in order to express the appropriate genetic program. However, since the number of environmental conditions is much larger than that of available genetic programs so that the cell may utilize the same genetic program for a large set of conditions, it may not have evolved a signaling pathway for every environmental condition, notably those that are rarely encountered. Here we show that in the absence of signal transduction, switching to the appropriate attractor state expressing the genes that afford adaptation to the external condition can occur. In a synthetic bistable gene switch in Escherichia coli in which mutually inhibitory operons govern the expression of two genes required in two alternative nutritional environments, cells reliably selected the “adaptive attractor” driven by gene expression noise. A mathematical model suggests that the “non-adaptive attractor” is avoided because in unfavorable conditions, cellular activity is lower, which suppresses mRNA metabolism, leading to larger fluctuations in gene expression. This, in turn, renders the non-adaptive state less stable. Although attractor selection is not as efficient as signal transduction via a dedicated cascade, it is simple and robust, and may represent a primordial mechanism for adaptive responses that preceded the evolution of signaling cascades for the frequently encountered environmental changes

A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (http://partsregistry.org/)