Searching for DNA–protein Interactions by Lambda Phage Display

Cicchini, Carla; Ansuini, Helenia; Amicone, Laura; Alonzi, Tonino; Nicosia, Alfredo; Cortese, Riccardo; Tripodi, Marco; Luzzago, Alessandra

doi:10.1016/s0022-2836(02)00851-3

Cited by 35 publications

(21 citation statements)

References 21 publications

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“…For given target DNA sites, phage display has emerged as a powerful tool, for example, for selecting transcription factors that recognize the given target out of millions of protein variants [5]. To study the eVects of individual protein mutants on the interaction, other methods that permit analysis of only one interaction at a time, such as gel mobility shift analysis [6], Southwestern blotting [5], enzyme-linked immunosorbent assay (ELISA) 1 -based methods [7], and Biacore analysis [6,7], had to be used.…”

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confidence: 99%

“…To study the eVects of individual protein mutants on the interaction, other methods that permit analysis of only one interaction at a time, such as gel mobility shift analysis [6], Southwestern blotting [5], enzyme-linked immunosorbent assay (ELISA) 1 -based methods [7], and Biacore analysis [6,7], had to be used. However, these methods are costly and are limited by low throughput.…”

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confidence: 99%

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Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten

Possling

Blaesing

et al. 2004

Analytical Biochemistry

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To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-aYnity DnaA box (R4) and with a low-aYnity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433-442 of Escherichia coli DnaA. This fragment contains six residues that were identiWed as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.  2004 Elsevier Inc. All rights reserved.

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confidence: 99%

mentioning

confidence: 99%

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten

Possling

Blaesing

et al. 2004

Analytical Biochemistry

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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%

“…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). We asked which vector would efficiently display Max bHLHZip and allow its binding to a natural dimerization partner, the GST fusion protein Mad (2).…”

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

confidence: 99%

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 in vitro methods for detecting protein-DNA interactions have been established, including ELECTROMOBILITY SHIFT ASSAYS, ENZYMELINKED IMMUNOSORBENT ASSAY (ELISA)-based methods or phage-display approaches, which have led to the identification of several interaction partners for one target 87 . Phage display has emerged as a powerful tool for selecting a protein that recognizes the DNA target from among millions of protein variants 88 . However, such in vitro selection methods are time consuming, have a limited throughput and often only the strongest interaction is selected.…”

confidence: 99%

Miniaturization in functional genomics and proteomics

et al. 2005

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| Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future. SYSTEMS BIOLOGYThe study of the complex interactions that occur at all levels of biological information -from whole-genome sequence interactions to developmental and biochemical networks -and their functional relationship to organism-level phenotypes. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 465 and concomitantly reducing the complexity of genomic DNA. Although PCR can be used to amplify small numbers of DNA molecules in low microlitre or nanolitre volumes in vitro, problems are associated with the dilution of heterozygous DNA. This could result in incorrect genotyping results (homozygous genotypes) because of the preferential amplification of one of the alleles. Extreme dilution indicates that there is a sufficiently high integrity of the nucleic-acid templates to allow efficient amplification. Furthermore, amplification of low concentrations of nucleic acids requires strict quality control, as it is often associated with contamination problems, which can be problematic in high-throughput applications. In contrast to nucleic-acid manipulation, it is currently impossible to amplify proteins from a biological sample. R E V I E W S Miniaturization in functional genomicsGenome research and functional genomics have catalysed the development of high-throughput and miniaturized approaches for the analysis of biomolecules [5][6][7][8] . Figure 1 | DNA micorarrays. DNA microarrays that are based on non-porous solid supports such as glass have paved the way for highly parallel miniaturized analysis of biomolecules. Using robotic workstations, ~0.25-1 nl of DNA solution are printed on a slide, creating spots that range from 100 to 150 µm in diameter. Successive samples are then spaced to avoid contact between adjacent spots, with approximately 200 µm between each spot 7 . Currently, about 50,000 cDNAs can be robotically spotted onto a microscope slide and hybridized with a fluorescently labelled probe. Using photolithographical masking techniques, arrays that have 400,000 distinct oligonucleotides have been produced, each in its own 20-µm 2 region (today, this can be as small as ~5 µm 2 ) REF. 8. After spotting, nucleic-acid samples can be hybridized on these pre-structured microarrays. Hybridization events are monitored using fluorescence scanners. A typical fluorescence read-out of highly parallel hybridization on a cDNA microarray is shown in the bottom panel. Box 1 | Technological problems in miniaturizationEvery molecular biological method can be miniaturized from microl...

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Searching for DNA–protein Interactions by Lambda Phage Display

Cited by 35 publications

References 21 publications

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

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

Miniaturization in functional genomics and proteomics

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