Sort Of Library Research Articles

Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments

Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10 6 and 10 7 are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288–294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naïve human Fab yeast display library of over 10 9 clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5×10 9 clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.

Multiplexed sorting of libraries on libraries: a novel method for empirical protein design by affinity-driven phage enrichment on synthetic peptide arrays.

Chemically synthesized peptide arrays on planar cellulose carriers are proposed as libraries of ligands suitable for the multiplexed simultaneous capture of peptide-specific acceptor proteins from a large randomly mutagenized library of acceptor proteins presented on bacteriophage M13 particles. This experimental set-up can be exploited to rapidly screen for individual new, distinct binding partners from two complementary libraries (two-dimensional screening). The technical feasibility of this empirical protein design approach was demonstrated with calmodulin as an aceptor protein using an array of mastoparan variants for multiplexed phage affinity enrichment.

Engineering Antibody Affinity by Yeast Surface Display

This chapter elaborates the engineering antibody affinity by yeast surface display (YSD). YSD is a powerful tool for engineering the affinity, specificity, and stability of antibodies, as well as other proteins. The methods for displaying an antibody on yeast, creating mutant libraries and sorting libraries for improved clones are presented. YSD involves the expression of a protein of interest on the yeast cell wall, where it can interact with proteins and small molecules in solution. The protein is expressed as a fusion to the Aga2p mating agglutinin protein, which is in turn linked by two disulfide bonds to the Aga1p protein linked covalently to the cell wall. Labeling yeast that are displaying an antibody or antibody library with a fluorescent or biotinylated antigen allows quantification of binding affinity, and enables library sorting by fluorescence-activated cell sorting. A second fluorophore conjugated to an antibody is used to detect the epitope tag C-terminal to the scFv, which allows for the normalization of expression and eliminates nondisplaying yeast from quantification. It is found that a complete cycle of mutagenesis and screening, from wild-type clone to improved mutant clone requires conservatively approximately 3–6 weeks.

Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.

Multiplexed Affinity Sorting and Analysis of Libraries on Libraries: Applications to Protein Design and Proteome Research

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Country House Library Reassess'd: or, Did The ‘Country House Library’ Ever Really Exist?

Most of us think we know a country house library when we see one, but in reality surprisingly little work has actually been done on them. As the Trust's collection-level survey programme is starting to show, many of the favourite commonplaces are misleading or entirely mythical. If we are to get to grips with country house libraries, we must recognize them for what they were — just one sort of private library. But above all we must look at their books.

Recent advances in the molecular genetics of Paramecium.

Paramecium continues to be used to study motility, behavior, exocytosis, and the relationship between the germ and the somatic nuclei. Recent progress in molecular genetics is described. Toward cloning genes that correspond to mutant phenotypes, a method combining complementation with microinjected DNA and library sorting has been used successfully in cloning several novel genes crucial in membrane excitation and in trichocyst discharge. Paramecium transformation en masse has now been shown by using electroporation or bioballistics. Gene silencing has also been discovered in Paramecium, recently. Some 200 Paramecium genes, full length or partial, have already been cloned largely by homology. Generalizing the use of gene silencing and related reverse-genetic techniques would allow us to correlate these genes with their function in vivo.

Ready for the information society? A study of Cape Town's children's librarians

Sixty‐seven children's librarians were interviewed with a view to assessing their readiness to take on an enhanced educational role as information literacy educators. If South Africa is to compete in the global economy of the 21st century, we need information literate school leavers equipped for lifelong learning, yet less than a third of our schools has any sort of library. The study finds virtual unanimity that the introduction of the new South African curriculum has brought increased pressure ‐ with virtually all respondents commenting that they are doing the work of teachers. But it reveals that the librarians lack capacity in terms of their skills and their facilities. There is consensus among them that their needs are not being met and that their services to school children are inadequate.

A Novel Soluble Tissue Factor Variant with an Altered Factor VIIa Binding Interface

Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa). To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein. TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa. Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence. Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface. The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58. Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation. Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.

Kunitz domain inhibitors of tissue factor-factor VIIa. II. Potent and specific inhibitors by competitive phage selection.

Active-site inhibitors of the human tissue factor-Factor VIIa complex (TF.FVIIa) that are both potent and specific have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries using a strategy termed competitive phage selection. Phage display Libraries I-III, previously sorted by direct selection, were sorted by competitive selection against immobilized TF.FVIIa in which increasing amounts of Factor XIa (FXIa) were added to the selection buffer to remove phage with high affinity for FXIa. The most striking difference in the selected Kunitz domain sequences was at the P4' position where Lys was highly preferred instead of Leu which was found here by direct selection (Dennis, M. S., and Lazarus, R. A. (1994) J. Biol. Chem. 269, 22129-22136). In addition, both Lys and Arg were selected in the P1 position as opposed to Arg alone. A new library (Library IV) was constructed which contained all previously observed amino acids at 9 positions in the primary and secondary binding loops of APPI. Comparable results were obtained by sorting Library IV against immobilized TF.FVIIa in the presence of either FXIa alone or FXIa, plasma kallikrein, and plasmin. The Kunitz domains obtained by either competitive selection strategy potently inhibited TF.FVIIa, with Ki* values ranging from about 2 to 20 nM; the Ki* values were generally > 1 microM for FXIa and plasma kallikrein and ranged from 4 to 200 nM for plasmin. Variants selective for TF.FVIIa were assayed for free FVIIa and FXa inhibitory activity and characterized in coagulation assays. A rationale for the selection of Lys at both the P4' and P1 positions based upon comparison of sequences and structures of relevant serine proteases and inhibitors is presented.

Rapid physical mapping of YAC inserts by random integration of I-Sce I sites.

We have developed a novel strategy, based on the random insertion by homologous recombination of artificial I-Sce I sites within mammalian repetitive DNA sequences, which should greatly facilitate the high resolution physical mapping of large DNA fragments cloned in YAC. A set of transgenic yeast strains containing appropriately spaced I-Sce I sites within the YAC insert defines a series of nested physical intervals against which new genes, clones or DNA fragments can be mapped by simple hybridisation. Sequential hybridisation using such a series of nested YAC fragments as probes can also allow the rapid sorting of phage or cosmid libraries into contigs. This approach, which has been applied to a YAC containing a 460 kb insert from the mouse X chromosome, may also have applications for the restriction mapping of large genomic segments, mapping of exons and the search for homologous genes.

Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes.

We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.

Analysis of the 5′-AAUAAA motif and its flanking sequence in human RNA: relevance to cDNA library sorting

The motif, N- 8..N- 1AAUAAAN 1..N 8 (where N is A, C, G or U), and its flanking sequence in human mRNA were examined by database analysis. Approximately 20% of 5′-AAUAAA in 3′-noncoding regions appear not to direct mRNA cleavage-polyadenylation. In coding regions, Asn-Lys, Ile-Lys and Ile-Asn are proven not to be unfavourable, and AAUAAA is not an unfavourable choice of coding sequence, occuring in 16% of mRNAs. Neither immediate flanking sequence nor associated motifs bear sufficient information content to account for the cleavage specificity observed. The unusual distribution and properties of the motif, AATAAA, in cDNA invite novel strategies for sorting cDNA libraries.

Pesaro, fano, urbino: Libraries in three small cities of central Italy

Italy abounds in libraries, private and public. Within the system of forty-seven libraries regulated by the state are the national central libraries of Rome and Florence; the national libraries of Milan, Naples, Palermo, Bari, Turin and Venice; twelve university libraries; eleven libraries annexed to national monuments-Montecassino, for instance, and Subiaco; and sixteen special libraries, among them the Biblioteca Mediceo-Laurenziana, formed from the collections of Cosimo the Elder, Pietro de’ Medici, and Lorenzo the Magnificent. However, these libraries of the state public system, with their marvellously rich and varied holdings, are essentially the domain of scholars: they have about as much impact on the daily life of the ordinary Italian citizen as the Huntington Library in Pasadena has on the everyday life of the residents of Bakersfield, California. Readers in Bakersfield who want to find out how to build a patio, or win at small claims court or poker, or who crave a weekend’s worth of science fiction, go to the Kern County library or one ofits branches. What sort oflibrary service is available to the citizens of the Italian equivalent of Bakersfield? According to the UNESCO Statistical Yearbook, 1976, there are in Italy 8686 municipal libraries serving 2 944 163 registered borrowers. Curious to know something of the day-to-day operation of Italian municipal libraries outside major metropolitan areas, and of the concerns of their librarians, I chose the province of Pesaro-Urbino, in the region Le Marche of central Italy, as my field of investigation. Pesaro-Urbino, 1117 square miles in area, with 330000 people, is similar to Kern County in population, and similar as well in that it is predominately agricultural: 42% of its residents work in agriculture; 34% in industry. At this point the similarities cease, for Pesaro-Urbino enjoys forty-two kilometers of Adriatic coastline dotted with beach resorts which draw huge numbers of Italian and European tourists each summer. This narrow coastal plain rises abruptly to the foothills of the

Juana de Laban Collection

It is a modest space, an angular, cramped room with exposed pipes overhead, a concrete floor. On all sides sprout wooden shelves laden with three thousand books and journals, photographs, slides, programs, and memorabilia – the Juana de Laban Collection, now housed at the Dallas Theater Center. To find the collection, one must climb narrow stairs and burrow through the Theater's wardrobe area to the tiny room beyond that houses the material lovingly collected by that bright and fiercely proud dance historian and critic who was also, overwhelmingly, the daughter of Rudolf Laban.Any scholar's library reflects his heart and mind, his interests and aspirations, his education. Juana de Laban's collection reveals an intellect remarkably catholic in its taste, astonishing in its breadth. It is the sort of library that skips across disciplines to include volumes on anthropology, psychology, history, aesthetics and criticism, all related to the study of human movement.

Technical College libraries: 1 Problems of classification

Almost every college with any sort of library must by now have found it necessary to arrange its books and pamphlets in some sort of subject order.

Polyphase Merge Sorting - An Advanced Technique

Designers of generalized library sort packages for the current and future generations of computers are faced with the challenge of developing new techniques that provide more effective use of these computers. The major concern in developing efficient sorting routines in the past has been the internal sorting techniques, that is, the methods of manipulating the data within the memory of the computer. Precise methods must, of course, be devised for each new computer design but, due to the extensive effort in this area in the past, few new internal sorting techniques have been introduced for, what are in computer terms, generations.

Library Development in the Post‐war Period: a Symposium

Readers are aware of the fact that the Council of the Library Association has circulated a pamphlet, outlining its views and proposals regarding post‐war arrangements under library authorities. This pamphlet has been much discussed by committees in many places. With a view to assisting readers to clarify their views on the subject, we arranged for this symposium, the contributors to which represent every sort of library under public authority. We would be pleased to publish further contributions from readers who have views on the subject.

LIBRARIES FOR THE PATIENTS IN HOSPITALS FOR THE INSANE

Some sort of medical library in the modern hospital for the insane is an understood thing and must be carried on along fairly conventional lines, but the organized central library for the use of the patients is a comparatively new idea and presents several problems peculiar to itself. In order to find out how the various hospitals are meeting these problems and how far the organized library is superseding the old plan of bookcases on the wards, a circular letter was sent from McLean Hospital to 120 of the largest and most representative institutions of the kind in this country and in Canada. Answers were received from 95; adding for statistical purposes McLean itself, we have a basis of 96 hospitals and asylums from which to draw our conclusions. Of these 96, there are only 15 which have no books at all. The superintendents of several of these latter hospitals deplore the fact, most of them giving the illiteracy or the destructiveness of their patients as the cause; one speaks of lending his own books ; two frankly state that they do not believe in libraries in such institutions. Twenty-one more hospitals have no central library, but report books on the various wards ranging in numbers from 50 to 5000 volumes. Several of these superintendents write

The British Museum Library

WTHAT sort of reference library can be provided in connection with the natural history collections when they are moved from the British Museum to South Kensington? is a subject now under consideration. It is stated ol good authority that, so far as the building arrangements at Kensington go, no provision whatever has been made for library space, and that in the Act passed at the end of last session to enable the trustees to move the collections, a reference library seems to have been entirely overlooked. That Act has, however, been the subject of a resolution by the General Committee of the British Association, requesting the Council to take such steps in the matter as they might deem expedient; and although the resolution had principal reference to the administration of the collections, its force extends equally to such an important matter as a library, should the Council “deem it expedient” to include that subject.