Toward selection of internalizing antibodies from phage libraries

Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).     

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.     

Selection and characterization of lipase abzyme from phage displayed antibody libraries

Antibodies with enzymatic activity were named abzymes or catalytic antibodies. In the present study, the lipolytic abzymes were selected from the phage displayed antibody libraries against a transition state analog (TSA) of lipases/esterases. After three rounds of selection, four monoclonal phage particles capable of binding significantly with the TSA were obtained. The soluble scFv antibody fragments were further expressed and obtained using Escherichia coli strain HB2151. The binding capabilities and the apparent enzymatic activities of the purified antibody proteins were measured. The 3D structures of the expressed antibodies were also predicted through homology modeling and binding-site prediction algorithm. The present method demonstrates that selection from phage displayed antibody libraries is an efficient and convenient means to find new abzymes.     

In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.     

In vitro selection using a dual RNA library that allows primerless selection

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC₅₀ of 4.5 nM at 37°C.     

In vitro selection and characterization of HIV-1 variants with increased resistance to sifuvirtide, a novel HIV-1 fusion inhibitor

Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide.     

Large scale production of phage antibody libraries using a bioreactor

One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.     

In vivo selection of RNA-binding peptides from combinatorial libraries.

We have used a two step procedure to identify peptides that bind strongly to the Rev-response element (RRE) of HIV. In the first step, RRE-binding peptides were screened from a combinatorial peptide library generated by "randomization" using a small subset of the 20 amino acids. In the second step, one such RRE-binding peptide, RSG-1, was "evolved" into an even stronger RRE-binding peptide using a codon-based mutagenesis procedure. After 2 rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity than wild-type Rev peptide.     

In vitro selection of Nicotiana sylvestris variants with limited resistance to TMV

Haploid tobacco (Nicotiana sylvestris) plants were inoculated with a yellow strain of tobacco mosaic virus (TMV-Flavum) and then exposed to 500 rads of acute gamma radiation. Leaf strips cultured on callus-inducing medium yielded two types of colonies: 1) yellow, virus-infected and 2) green, apparently healthy. Of the 3210 calli scored, approximately 5% were virus-free, and after regeneration, 0.2% were resistant at the plant stage. Later, adult plants, both TMV-resistant and TMV-susceptible, produced self-fertile, diploid flowers. Both seedling progeny and rooted cuttings from resistant stock plants showed resistance to TMV infection. This resistance was characterized by restricted virus multiplication and movement within the infected plant resulting in a 3–8 week delay in symptom expression.Journal Paper No. 10138 of the Michigan Agricultural Experiment Station.This work was supported in part by U.S.D.A. grant no. 79-59-2261-1-1-351-1.We thank Drs. M. Daub, R. Griesbach, and J. Hunsperger for helpful suggestions.The excellent technical assistance of Ms. Brenda Floyd and Sara Stadt is acknowledged.     

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.     

In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries

The covalent coupling of an mRNA to the protein that it encodes (mRNA display) provides a powerful tool for analysis of protein function in the post-genomic era. This coupling allows the selective enrichment of individual members from libraries of displayed proteins and the subsequent regeneration of an enriched library using the RNA moiety. Tissue-specific libraries from poly(A)(+) mRNA were prepared by priming first and second strand cDNA synthesis with oligonucleotides containing nine random 3' nucleotides, the fixed regions of which encoded the requisite sequences for formation of mRNA display constructs and a library-specific sequence tag. Starting with a pool of uniquely tagged libraries from different tissues, an iterative selection was performed for binding partners of the anti-apoptotic protein Bcl-X(L). After four rounds of selection, the pool was deconvoluted by polymerase chain reaction amplification with library-specific primers. Subsequent clonal sequence analysis revealed the selection of three members of the Bcl-2 family known to bind to Bcl-X(L). In addition, several proteins not previously demonstrated to interact with Bcl-X(L) were identified. The relative binding affinities of individual selected peptides were determined, as was their susceptibility to competition with a BH3 domain peptide. Based on these data, a putative BH3 domain was identified in most peptides.     

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel.     

Identification of a novel calpain inhibitor using phage display

Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.     

In vitro selection of Jun-associated proteins using mRNA display

Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein–protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein–protein interactions and can contribute to the comprehensive mapping of protein–protein interactions.     

In vitro selection of glyphosate-tolerant variants from long-term callus cultures of Zoysia matrella [L.] Merr

A complete protocol of in vitro selection and greenhouse screening for glyphosate-tolerant variants in manilagrass (Zoysia matrella [L.] Merr) was established in this study. Newly subcultured calli of more than 5?years?? old were transferred to selection medium containing 2?mM glyphosate. After two rounds of selection, 220 calli survived out of 840 and were transferred to regeneration medium without glyphosate. Regenerated plantlets were then transferred to regeneration medium containing 0.5?mM glyphosate to select tolerant plantlets. After 1-month growth, there were plantlets remained green and new shoots formed beside or on discolored explants. These surviving organisms were then transferred to fresh regeneration medium for further growth. Fully developed plantlets were transferred to a green house and then subjected to greenhouse screening by foliar spraying with 0.05?% glyphosate solution. Six glyphosate-tolerant plantlets, TP1-TP6, were obtained and proliferated for determination of sod-tolerance using morphological and physiological measurements. Fourteen days after foliar application with 0.1?% glyphosate, only TP5 showed enhanced sod-tolerance. The dark green color index value of TP5 was significantly higher than CK2, demonstrating that TP5 suffered less injury from glyphosate than CK2. Different physiological characters were also observed in CK1, CK2 and TP5. Significantly higher chlorophyll a content and catalase activity were observed in TP5 than in CK1. Fourteen days after treatment (DAT), the ion leakage, proline content and ascorbate peroxidase activity of CK2 and TP5 increased significantly, but the ion leakage of TP5 was significantly lower than that of CK2. The guaiacol peroxidase activity of TP5 increased significantly 14 DAT, and was significantly higher than that of CK1 and CK2. No change in shikimate content was observed in CK2 or TP5 14 DAT.