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Bioinspired Nanomaterials for Improving Sensing and Imaging Spectroscopy
Published in Kaushik Pal, Nanomaterials for Spectroscopic Applications, 2021
Janti Qar, Alaa A. A. Aljabali, Tasnim Al-Zanati, Mazhar S. Al Zoubi, Khalid M. Al-Batanyeh, Poonam Negi, Gaurav Gupta, Dinesh M. Pardhi, Kamal Dua, Murtaza M. Tambuwala
Targeting peptide molecules can be chosen primarily in three separate ways [34]; (2) chemical synthesis and structure-based computational engineering and design [11, 43]; and (3) screening of peptide libraries [43]. A phage display is a conventional method, widely used because of its benefits such as easy handling and multiple different peptides that can be screened appropriately [18], on the basis of a select peptide which connects with the desired target from a phage library [45]. Every phage clone shows one peptide, and up to 10 peptides can be displayed in the entire library. So unbound phages have been washed, and people with required binding behavior are retrieved and eventually restudied through competitive elution. There are several methods of affinity screening to improve the probability of receiving peptides with a strong affinity with the targeted molecules [18]. Mostly these recognition measures were subject to in vivo molecular imaging chemical and biological assessments.
Engineering Living Materials: Designing Biological Cells as Nanomaterials Factories
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Peter Q. Nguyen, Pichet Praveschotinunt, Avinash Manjula-Basavanna, Ilia Gelfat, Neel S. Joshi
Besides bacteria, viruses such as the filamentous bacteriophage M13 are also employed to bind to inorganic nanomaterials (Kehoe and Kay 2005; Wen and Steinmetz 2016). Wild-type M13 is 6.5 nm in diameter and 930 nm in length. A typical M13 virion is covered with 2700 copies of the major coat protein (pVIII) and 5 copies of minor coat proteins (pIII, pVI, pVII and PIX). pIII and pVI form the one end of the virion, while pVII and pIX are at the other end. Peptides/proteins having affinity to a specific material are typically selected from a phage display library. In one particular example, a 15-mer peptide library displayed on the filamentous phage was used to investigate the crystallization of calcium carbonate (Li, Botsaris, and Kaplan 2002). The template effect of the phage-displayed peptides resulted in hollow spheres of calcium carbonate nanoparticles (Figure 8.2c). Interestingly, the bacteriophage was found to slow down the phase transformation from vaterite to calcite.
Overview of the Manifold VNPs Used in Nanotechnology
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Phage display technology. Besides its manifold applications in materials, M13 has long been exploited as a platform for phage display technologies. Phage display is a high-throughput screening technique that is used to identify peptides that are specifically interacting with molecular proteins or receptors (Arap et al., 2002; Hajitou et al., 2006; Nanda & St. Croix, 2004; Ruoslahti, 2002) or even synthetic materials (for a review see Flynn et al., 2003). In brief, DNA sequences encoding for random peptide sequences are ligated into the pIII or pVIII gene. The peptides are thus displayed on the surface of the phages. The phage is then produced in E. coli. The target (protein or inorganic material) is immobilized on a surface and phages are applied. The unbound phages are rinsed off; those that are bound to the target are then eluted and amplified in the expression system. This cycle of binding, elution, and amplification is called biopanning and repeated several times to select the phages with highest selectivity and specificity. The amino acid sequence of the desired peptide is readily obtained by sequencing of the encoding nucleic acid of the selected phage.
Characterization of the novel anti-TNF-α single-chain fragment antibodies using experimental and computational approaches
Published in Preparative Biochemistry and Biotechnology, 2019
Samira Pourtaghi-Anvarian, Samin Mohammadi, Maryam Hamzeh-Mivehroud, Ali Akbar Alizadeh, Siavoush Dastmalchi
Phage display technology is a combinatorial biology approach in which a library of phage particles displaying peptides, proteins, and antibodies is used to isolate specific antigen binders.[20] In this technology, highly diverse libraries allow rapid identification and isolation of specific ligands for any targets such as enzymes and cell surface receptors.[21] Structural analysis of selected ligands could provide new insights into ligand-target interaction useful in drug discovery and development. In our previous study, three phage displaying scFv antibodies (i.e. J43, J44, and J48) capable of binding to TNF-α were isolated from Tomlinson antibody libraries using phage display technology.[13] The current investigation aimed to produce and purify these scFvs for further evaluation of their binding characteristics to TNF-α. DNA sequencing of the identified scFv antibodies showed the existence of amber stop codons in their coding sequences preventing straightforward expression of these scFvs devoid of pIII phage coat protein in host bacteria. To solve this problem, a pair of overlapping primers was used to mutate the stop codons into normal amino acids codons (Figure 1) and the generated DNA sequences were cloned into pIT2 phage vector. The coding sequences of scFvs in pIT2 phage vector are followed by 6 × His tag DNA sequence, enabling expression of these antibodies as His-tagged fusion proteins. Phages displaying scFvs were infected into E.coli HB2181 expression system and the production of the corresponding proteins was induced by 1 mM IPTG. The produced scFvs were purified on an affinity column and analyzed on SDS-PAGE (Figure 2A). The purity of the obtained scFvs was assessed by semi-quantification of the SDS-PAGE gel using mage J program resulting in about 85% purity for all three scFvs. The expression of the His-tagged scFvs was confirmed using ECL in Western blotting technique and the results are shown in Figure 2B.