Eficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda

Santini, Claudia; Brennan, Debra; Mennuni, Carmela; Hoess, Ronald H.; Nicosia, Alfredo; Cortese, Riccardo; Luzzago, Alessandra

doi:10.1006/jmbi.1998.1986

Cited by 84 publications

(57 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Greater success appears to have been achieved with -based vectors for cDNA display (Santini et al 1998;Beghetto et al 2001). However, even though such C-terminal intracellular vectors increase the likelihood that ORFs will be displayed, they do not per se provide any selective pressure for ORFs.…”

mentioning

confidence: 99%

Selecting Open Reading Frames From DNA

Zacchi

Sblattero²,

Florian³

et al. 2003

Genome Res.

View full text Add to dashboard Cite

We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the β-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the β-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems

show abstract

mentioning

confidence: 99%

Selecting Open Reading Frames From DNA

Zacchi

Sblattero²,

Florian³

et al. 2003

Genome Res.

View full text Add to dashboard Cite

show abstract

“…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%

See 1 more Smart Citation

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

Ciarapica

Rosati

Nasi

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

“…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.…”

mentioning

confidence: 99%

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

Vı́lchez¹,

Jacoby

2004

Appl Environ Microbiol

View full text Add to dashboard Cite

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...

show abstract

Eficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda

Cited by 84 publications

References 31 publications

Selecting Open Reading Frames From DNA

Selecting Open Reading Frames From DNA

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

Contact Info

Product

Resources

About