Antibody library selection by the β-lactamase protein fragment complementation assay

Secco, Paola; D'Agostini, Elena; Marzari, Roberto; Licciulli, Marta; Niro, Roberto Di; D’Angelo, Sara; Bradbury, Andrew; Dianzani, Umberto; Santoro, Claudio; Sblattero, Daniele

doi:10.1093/protein/gzn053

Cited by 17 publications

(18 citation statements)

References 66 publications

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“…Although it is considered to be highly specific, it generally requires confirmation of the binding using alternative methods. In our case, we used the PCA, a method previously used in our laboratory (26). All the selected TG2 interactors were cloned into the pω vector, in frame with the C-terminal fragment (aa 196–286) of TEM-1 β-lactamase, while the human TG2 gene was cloned into the pα vector, fused to the α fragment (aa 1–195, N-terminal).…”

Section: Resultsmentioning

confidence: 99%

“…Details for pα and pω vector construction are described in (26). The DNA fragments of interactors were cut from pPAO10 with BssHII and NheI restriction enzymes, and ligated into the pω vector.…”

Section: Methodsmentioning

confidence: 99%

“…The TG2 gene was digested with EcoRI and HindIII, and ligated into the pα vector. As positive control, co-transformed bacteria pα-RON2/pω-sc7 were used according to Secco et al (26). pω-ΔG2 was used as negative control for the interaction with TG2: ΔG2 is a scFv recognizing a portion of the Cholera toxin (DG).…”

Section: Methodsmentioning

confidence: 99%

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Rapid interactome profiling by massive sequencing

Niro

Sulic

Mignone

et al. 2010

Nucleic Acids Research

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We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Rapid interactome profiling by massive sequencing

Niro

Sulic

Mignone

et al. 2010

Nucleic Acids Research

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“…Another consequence of the burgeoning genomic data is the absence of suitable analytical reagents such as antibodies to investigate the predicted proteome, due to the low‐throughput of conventional antibody development protocols. Many groups have developed novel technologies to address this situation, involving combinatorial yeast‐phage display, selectively infective phage, protein fragment complementation assays (PCAs) and selection by avidity capture (SAC) 10–13. These techniques rely on simultaneous combinatorial expression of antigen and antibody libraries and conditional enzymatic activity or fluorescence‐activated cell sorting (FACS) for selection of cognate pairs.…”

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confidence: 99%

The body donation program at the Federal University of Health Sciences of Porto Alegre: A successful experience in Brazil

Rocha

Tormes

Lehmann

et al. 2012

Anatomical Sciences Ed

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The use of dissection to study human anatomy is the foundation for educational excellence among future health professionals, as it offers an ideal opportunity to learn the body's morphology in three dimensions while also providing students with a more humanistic education. The shortage of bodies for dissection, combined with the Brazilian population's lack of knowledge concerning the possibility of voluntarily donating their own bodies, led to the creation of the Body Donation Programs for Education and Research in Anatomy at the Federal University of Health Sciences of Porto Alegre (UFCSPA). The program is based on three pillars: Informing the general public about the program, donor registration, and donation itself. Since the creation of the donor program in 2008, there has been an increase in both the number of donations made during donor's lifetime and the number of bodies received by the university. There has also been a shift in relation to the origin of these bodies, as before the creation of the program most bodies were unclaimed cadavers, while today most of the bodies are sourced from voluntary donations. The initial results regarding the public's acceptance of the possibility of making body donations have been encouraging, as shown by the annual growth in donor registrations. Consequently, the quality and quantity of the material available for educational purposes have greatly improved.

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“…Coselection of cognate antibody-antigen pairs from combinatorial libraries has been attempted by using selectively infective phage (10,11) or protein fragment complementation (12)(13)(14) with only limited success. The central difference in our approach is that we use two different display platforms for the antibody and the antigen libraries.…”

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confidence: 99%

Libraries against libraries for combinatorial selection of replicating antigen–antibody pairs

Bowley

Jones²,

Burton³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen-antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen-antibody clones in a single experiment.antibody libraries ͉ human genome ͉ phage display ͉ yeast display T he generation of antibodies to scientifically and clinically important protein antigens has occupied researchers for the past 25 years and has led to the establishment of combinatorial antibody libraries (1-6). Essentially, these libraries constitute a synthetic immune system. Nowadays, such libraries are routinely prepared and contain antibody collections that exceed the diversity of natural repertoires by many orders of magnitude. These libraries are not restricted by tolerance, they avoid the use of live animals, and have yielded important therapeutic antibodies (6). The libraries are most often formatted in yeast (7,8) or phage (1, 4) so that single binding events can be replicated and high-affinity antibodies can be selected. However, we have yet to extract the full potential of these powerful library methods because we still select antibodies one antigen at a time (9).The bottleneck imposed when antibodies are selected to one antigen at a time is illuminated by the opportunities posed by the human and other genome projects. These projects have provided an explosion in the numbers of known proteins, and it would be desirable to generate a set of high-affinity monoclonal antibodies to each of them so that ultimately one has a set of antibodies to every protein in the genome. Because combinatorial antibody libraries are not restricted by immunological tolerance, any selfor nonself-protein can be bound by a member of the antibody library. This means that, with respect to a given antibody library, the human proteome can be considered to be a collection of antigens.The present article describes a solution to the problem of simultaneous selection of monoclonal antibodies to a large set of antigens rather than to one antigen at a time.Coselection of cognate antibody-antigen pairs from combinatorial libraries has been attempted by using selectively infective phage (10, 11) or protein fragment complementation (12-14) with only limited success. ...

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Antibody library selection by the β-lactamase protein fragment complementation assay

Cited by 17 publications

References 66 publications

Rapid interactome profiling by massive sequencing

Rapid interactome profiling by massive sequencing

The body donation program at the Federal University of Health Sciences of Porto Alegre: A successful experience in Brazil

Libraries against libraries for combinatorial selection of replicating antigen–antibody pairs

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