Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics 1 1Edited by J. M. Thornton

Santi, Elisabetta; Capone, Stefania; Mennuni, Carmela; Lahm, Armin; Tramontano, Anna; Luzzago, Alessandra; Nicosia, Alfredo

doi:10.1006/jmbi.1999.3471

Cited by 69 publications

(48 citation statements)

References 46 publications

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“…The consequent application of functional enrichment and robotic-based screening will allow rapid generation of information in the field of functional genomics. However, it has to be mentioned that all display technologies based on biological systems will suffer from constraints limiting the system itself [31], and these apply also for the display of cDNA libraries [32]. Obvious limitations are related to the restricted codon usage of E. coli as host, the lack of post-translational modification [33] and to the toxicity of some gene products expressed in prokaryotic systems [34].…”

Section: Discussionmentioning

confidence: 99%

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

Kodzius¹,

Rhyner²,

Konthur

et al. 2003

CCHTS

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Abstract:We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinityselected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.

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Section: Discussionmentioning

confidence: 99%

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

Kodzius¹,

Rhyner²,

Konthur

et al. 2003

CCHTS

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“…Rox and Mad at positions 2,8,11,12,16,23,25, and 26 favored the same amino acids. Surprisingly, Max residues occurred at low frequency in the clones showing the highest binding affinity for Mad and Rox (Table II), with the only exceptions being Lys 4 (46%) and Asn 5 (53%), as if the Max Zip amino acid sequence was tuned to guarantee dimerization flexibility rather than strength (Fig.…”

Section: Display Of Max Bhlhzip Domain On Phage-to Identify the Most mentioning

confidence: 98%

“…2B (19)). At positions 11 and 12 only a few of the residues present in the repertoire were found in the selected domains; the preference for Cys 12 was stronger than for Met 11 (76 versus 53%). All possible amino acids were found at position 9, where glycine occurred with a 65% frequency, and it was strongly preferred by high affinity binders (domains 43M, 72I, 42I, 13I, 98M, 27M, 18I, and 43I).…”

Section: Display Of Max Bhlhzip Domain On Phage-to Identify the Most mentioning

confidence: 99%

“…HZip domain repertoires, we tested both filamentous phage vectors, successfully exploited for the construction of peptide or antibody repertoires (9,10), and phage, reported to be generally more suitable for exposing large polypeptides (11)(12)(13). The DNA sequence encoding Max bHLHZip was cloned into the three filamentous phage vectors pC89, pC178, and pHEN⌬, to obtain N-terminal fusions to pVIII or pIII coat proteins (14,15) and into the display vector 4 (D4) to display fusions to the D-protein C terminus (8,16).…”

Section: Display Of Max Bhlhzip Domain On Phage-to Identify the Most mentioning

confidence: 99%

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Molecular Recognition in Helix-Loop-Helix and Helix-Loop-Helix-Leucine Zipper Domains

Ciarapica

Rosati

Nasi

2003

Journal of Biological Chemistry

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Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways.

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“…Several combinatorial methods are now available to create large libraries of mutant proteins that can be searched for toxins with higher potency and different specificities. However, the rate-limiting step is the selection of valuable proteins from the mutant pool.One way to overcome this problem is to display libraries of peptides or proteins on the surface of bacteriophages (23,28,29,34). This technique is particularly suitable for B. thuringiensis toxins due to the nature of Cry proteins.…”

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confidence: 99%

Display of Biologically Functional Insecticidal Toxin on the Surface of λ Phage

Vı́lchez¹,

Jacoby

2004

Appl Environ Microbiol

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The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.The bacterium Bacillus thuringiensis produces insecticidal proteins during sporulation. These toxins are expressed as protoxins that are packaged into crystalline inclusions. When ingested by susceptible insect larvae, the protoxin is solubilized by the unique environment of the host gut and activated by proteolytic cleavage with gut enzymes. The activated toxins are able to bind to receptor molecules present on the insect gut epithelium and insert into the membrane (6). This membrane penetration results in the formation of pores which cause colloid osmotic lysis and eventual insect death (17,32,38).Due to their receptor specificity, Cry proteins are only toxic to certain insects and are harmless to other organisms, including humans, other mammals, fish, and most beneficial insects (31). B. thuringiensis is one of the few microbes that have been successfully used in agricultural insect pest control. Liquid and powder formulation mixtures of spores and crystals from this bacterium have been used for more than 40 years on many crops. B. thuringiensis toxin genes have also been successfully expressed in plants, providing environmentally benign control of insect pests (11).Although in the past it was hoped that insects would not develop resistance to these toxins, insect resistance to B. thuringiensis toxins has been detected in insect populations, mainly in the laboratory, but also in the field (8, 36).One possible way of overcoming the problem of resistance is to generate novel toxins by genetic manipulation of B. thuringiensis genes. Several combinatorial methods are now available to create large libraries of mutant proteins that can be searched for toxins with higher potency and different specificities. However, the rate-limiting step is the selection of valuable proteins from the mutant pool.One way to overcome this problem is to display libraries of peptides or protei...

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Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics 1 1Edited by J. M. Thornton

Cited by 69 publications

References 46 publications

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

Molecular Recognition in Helix-Loop-Helix and Helix-Loop-Helix-Leucine Zipper Domains

Display of Biologically Functional Insecticidal Toxin on the Surface of λ Phage

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