Surface Of Filamentous Phage Research Articles

Vectorized Delivery of Alpha-GalactosylCeramide and Tumor Antigen on Filamentous Bacteriophage fd Induces Protective Immunity by Enhancing Tumor-Specific T Cell Response

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels, and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (α-GalCer), a lipid antigen-stimulating invariant natural killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/α-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of α-GalCer and a MHC class I restricted tumor-associated antigenic determinant to antigen-presenting cells via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free α-GalCer and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe, inexpensive, and versatile tool.

Recombinant antibodies targeting HIV-1 capsid protein (p24): the development of single-chain variable fragment

Event Abstract Back to Event Recombinant antibodies targeting HIV-1 capsid protein (p24): the development of single-chain variable fragment Siti Aisyah Mualif1* and Mohammad Tasyriq Che Omar2 1 School of Biomedical Engineering & Health Sciences, Faculty of Engineering, University of Technology Malaysia, Malaysia 2 University of Science, Malaysia, Malaysia Background Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS) disease. The increasing morbidity and mortality due to HIV/AIDS in the past decades has sparked an interest to combat HIV/AIDS. However, ongoing research against the HIV/AIDS pandemic has failed to completely eradicate the infection. Introduction of highly active anti-retroviral therapy (HAART) in early 1990s has reduced the death rate of HIV/AIDS, but it has also resulted in the development of drug-resistant strains of HIV. Therefore, there is a pressing need to develop new and improved therapeutic modalities. HIV-1 capsid protein (p24) plays important roles in both early and late stages of HIV-1 replication. Small molecule inhibitors and peptides targeting p24 have shown to inhibit viral infection. However, rapid clearance and toxicity are major drawbacks associated with the aforementioned therapeutic modalities. The potential of monoclonal antibodies (mAbs) targeting p24 was discovered and found out that p24-targeting antibodies can be developed into novel therapeutic modalities. However, natural or hybridoma-derived mAbs are large molecules and difficult to engineer. Recombinant DNA technology allows the engineering of antibodies in multiple formats. Therefore, the aim of this study was to generate, characterize, and evaluate recombinant antibodies targeting HIV-1 p24. Methods Recombinant anti-p24 antibodies were generated from a combinatorial library of variable domains cloned from a hybridoma cell line and subsequently expressed on the surface of filamentous bacteriophage. Recombinant scFvs reacting specifically with HIV-1 p24 were isolated, expressed, and characterized. Results Out of 50 clones, three specific binders were identified via initial ELISA screening. Specificity of the binders was confirmed through competition studies and the selected clones were expressed in E. coli. The recombinant scFvs markedly inhibited p24 polymerization in vitro and HIV replication in Jurkat T cell lines when expressed as intracellular antibodies (intrabodies). Conclusion The anti-p24 scFvs engineered in this study have potential to be developed into novel antibody-based therapeutics against HIV. Acknowledgements This work was supported by a FRGS grant (203/CIPPT/67 11206) from the Ministry of Higher Education of Malaysia and Research Student Fund (USM/IPPT/2000/G-2/xiv) from AMDI, USM. Not forgetting the MOHE scholarship (KPT (BS) 841003015520) for the author to complete the work and a PAS grant (PY/2017/01374) from UTM, Johor Bahru to support the presentation. Keywords: HIV-1 p24, scFv, Phage display antibody library, anti-p24 scFv, therapeutic Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Poster Presentation Topic: Infectious diseases Citation: Mualif S and Che Omar M (2019). Recombinant antibodies targeting HIV-1 capsid protein (p24): the development of single-chain variable fragment. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00104 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Siti Aisyah Mualif, School of Biomedical Engineering & Health Sciences, Faculty of Engineering, University of Technology Malaysia, Johor Bahru, Malaysia, aisyahmualif@utm.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Siti Aisyah Mualif Mohammad Tasyriq Che Omar Google Siti Aisyah Mualif Mohammad Tasyriq Che Omar Google Scholar Siti Aisyah Mualif Mohammad Tasyriq Che Omar PubMed Siti Aisyah Mualif Mohammad Tasyriq Che Omar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Selection of similar single domain antibodies from two immune VHH libraries obtained from two alpacas by using different selection methods

The two most used methods to select camelid single-domain antibody-fragments (VHHs) are: displaying their repertoires on the surface of filamentous bacteriophages (phage display) or linking them to ribosomes (ribosome display). In this study, we compared specific VHHs isolated from two different immune libraries coming from two different alpacas by using these two selection methods.Three anti-GFAP (glial fibrillary acidic protein) VHHs were derived from an immune library obtained by ribosome display after immunization of one alpaca with purified GFAP, a protein expressed by astroglial cells. In parallel, three other anti-GFAP VHHs were derived from an immune library by phage display after immunization of another alpaca with a human brain tissue extract containing GFAP. All the VHHs were closely related and one VHH was found to be strictly identical in both studies. This highlights the selection pressure exerted by the camelid immune system to shape the paratope of an antibody against a defined antigen.

Upregulation of Mrps18a in breast cancer identified by selecting phage antibody libraries on breast tissue sections

BackgroundOne of the hallmarks of cancer is an altered energy metabolism, and here, mitochondria play a central role. Previous studies have indicated that some mitochondrial ribosomal proteins change their expression patterns upon transformation.MethodIn this study, we have used the selection of recombinant antibody libraries displayed on the surface of filamentous bacteriophage as a proteomics discovery tool for the identification of breast cancer biomarkers. A small subpopulation of breast cells expressing both cytokeratin 19 and cytokeratin 14 was targeted using a novel selection procedure.ResultsWe identified the mitochondrial ribosomal protein s18a (Mrps18a) as a protein which is upregulated in breast cancer. However, Mrps18a was not homogeneously upregulated in all cancer cells, suggesting the existence of sub-populations within the tumor. The upregulation was not confined to cytokeratin 19 and cytokeratin 14 double positive cells.ConclusionThis study illustrates how phage display can be applied towards the discovery of proteins which exhibit changes in their expression patterns. We identified the mitochondrial protein Mrps18a as being upregulated in human breast cancer cells compared to normal breast cells.

Monoclonal Antibodies and Antibody Like Fragments Derived from Immunised Phage Display Libraries.

Morbidity and mortality associated with infectious diseases are always on the rise, especially in poorer countries and in the aging population. The inevitable, but unpredictable emergence of new infectious diseases has become a global threat. HIV/AIDS, severe acute respiratory syndrome (SARS), and the more recent H1N1 influenza are only a few of the numerous examples of emerging infectious diseases in the modern era. However despite advances in diagnostics, therapeutics and vaccines, there is need for more specific, efficacious, cost-effective and less toxic treatment and preventive drugs. In this chapter, we discuss a powerful combinatorial technology in association with animal immunisation that is capable of generating biologic drugs with high affinity, efficacy and limited off-site toxicity, and diagnostic tools with great precision. Although time consuming, immunisation still remains the preferred route for the isolation of high-affinity antibodies and antibody-like fragments. Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. The selection of binding fragments from phage display libraries has proven significant for routine isolation of invaluable peptides, antibodies, and antibody-like domains for diagnostic and therapeutic applications. Here we highlight the many benefits of combining immunisation with phage display in combating infectious diseases, and how our knowledge of antibody engineering has played a crucial role in fully exploiting these platforms in generating therapeutic and diagnostic biologics towards antigenic targets of infectious organisms.

The role of phage display in therapeutic antibody discovery.

Phage display involves the expression of selected proteins on the surface of filamentous phage through fusion with phage coat protein, with the genetic sequence packaged within, linking phenotype to genotype selection. When combined with antibody libraries, phage display allows for rapid in vitro selection of antigen-specific antibodies and recovery of their corresponding coding sequence. Large non-immune and synthetic human libraries have been constructed as well as smaller immune libraries based on capturing a single individual's immune repertoire. This completely in vitro process allows for isolation of antibodies against poorly immunogenic targets as well as those that cannot be obtained by animal immunization, thus further expanding the utility of the approach. Phage antibody display represents the first developed methodology for high throughput screening for human therapeutic antibody candidates. Recently, other methods have been developed for generation of fully human therapeutic antibodies, such as single B-cell screening, next-generation genome sequencing and transgenic mice with human germline B-cell genes. While each of these have their particular advantages, phage display has remained a key methodology for human antibody discovery due its in vitro process. Here, we review the continuing role of this technique alongside other developing technologies for therapeutic antibody discovery.

DNA-Encoded Chemical Libraries: Advancing beyond Conventional Small-Molecule Libraries

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

Automation Highlights from the Literature

Automation Highlights from the Literature

Tracking chemical reactions on the surface of filamentous phage using mass spectrometry

Chemical modification of phage libraries has allowed the in vitro evolution of ligands having properties not provided by natural polypeptides. The development of novel and more diverse chemical reactions on phage was hampered by the lack of analytical methods to efficiently monitor the reaction products on the more than 10 000 kDa large filamentous phage particles. Herein, we present a strategy to detect chemically modified peptides on phage based on enzymatic release of peptide from phage and mass spectrometry analysis.

New avenues for phage-display library to produce a Cryptococcus-specific anti-idiotypic antibody of HM-1 killer toxin

Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.

Design and Screening of M13 Phage Display cDNA Libraries

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.

Phage Display on the Base of Filamentous Bacteriophages: Application for Recombinant Antibodies Selection

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Phage Display on the Base of Filamentous Bacteriophages: Application for Recombinant Antibodies Selection

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Evaluation of humoral and cellular immune responses against HSV-1 using genetic immunization by filamentous phage particles: A comparative approach to conventional DNA vaccine

Phage display is based on expressing peptides as a fusion to one of the phage coat proteins. To date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. In recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as DNA delivery vehicles to mammalian cells. In this study, recombinant filamentous phage whole particles were used for vaccination of mice. BALB/c mice were inoculated with filamentous phage particles containing expression cassette of Herpes simplex virus 1 (HSV-1) glycoprotein D that has essential roles in the virus attachment and entry. Both humoral and cellular immune responses were measured in the immunized mice and compared to conventional DNA vaccination. A dose–response relationship was observed in both arms of immune responses induced by recombinant filamentous phage inoculation. The results were similar to those from DNA vaccination. Filamentous phages can be considered as suitable alternative candidate vaccines because of easier and more cost-effective production and purification over plasmid DNA or bacteriophage lambda particles.

Diversity and censoring of landscape phage libraries.

Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd-tet (landscape libraries). They were obtained by replacement of amino acids 2-4 and 2-5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space.

Filamentous Bacteriophage as a Novel Therapeutic Tool for Alzheimer's Disease Treatment

Antibodies towards the N-terminal region of the amyloid-beta peptide (AbetaP) bind to Abeta fibrils, leading to their disaggregation. We developed an immunization procedure using filamentous phages displaying the only four amino acids EFRH encompassing amino acids 3-6 of the 42 residues of AbetaP, found to be the main regulatory site for Abeta formation. Phages displaying EFRH epitope are effective in eliciting humoral response against AbetaP which, in turn, relieves amyloid burden in brains of amyloid-beta protein precursor transgenic mice, improving their ability to perform cognitive tasks. In order to overcome the low permeability of the blood brain barrier for targeting 'anti-aggregating' monoclonal antibodies (mAbs) to Abeta plaques in the brain, we applied antibody engineering methods to minimize the size of mAbs while maintaining their biological activity. Single-chain antibodies displayed on the surface of filamentous phage showed the ability to enter the central nervous system (CNS). The genetically engineered filamentous bacteriophage proved to be an efficient, nontoxic viral delivery vector to the brain, offering an obvious advantage over other mammalian vectors. The feasibility of these novel strategies for production and targeting of anti-aggregating antibodies against Abeta plaques to disease affected regions in the CNS may have clinical potential for treatment of Alzheimer's disease.

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

BackgroundThe ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies.ResultsAn enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.ConclusionThe construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

Translational applications of antibody phage display

Recent developments in molecular immunology have facilitated the expression of immune repertoires in the form of immunoglobulin fragments on the surface of filamentous bacteriophage. Such approaches, known as "phage display", have provided powerful tools for producing monoclonal antibodies for research, clinical, and therapeutic applications. Our laboratory has combined these techniques with novel selection methods to isolate extraordinarily large arrays of human antibodies that can be used for developing new diagnostic reagents and methods for their use, as well as reagents that may serve as leads for the design of novel therapeutic molecules. In particular, application of phage display to the study of antibody-mediated autoimmune disease, notably idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, and pemphigus have facilitated comprehensive genetic and serologic analyses of pathogenic autoantibodies thereby offering new insights into disease pathophysiology and approaches for their treatment.

Gene transfer of Hodgkin cell lines via multivalent anti-CD30 scFv displaying bacteriophage

BackgroundThe display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low.ResultsHere we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII.ConclusionWe can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors.

Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules.

Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters Km and kcat were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a Km of 11.2 µM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 µM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.