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Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-term...
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... using these coat proteins, N-terminal fusion of the target is mandatory for successful phage propagation. For the display of cDNA libraries, N-terminal fusion is not possible due to inherent stop codons present in the cDNA fragments, as represented in Figure 1. However, the free carboxyl terminus of minor coat protein 6 (p6) allows successful fusion of the cDNA without interfering with phage propagation [5]. ...
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... previous studies were based on N-terminal fusion to p3 or p8, frameshifting had to occur in order to express the p3 and p8 coat proteins that enable phage propagation. However, C-terminal fusion to p6 does not require expression of the inserted cDNA for successful phage propagation as represented in figure 1 [5,6]. Therefore, other methods must be used to detect the possible occurrence of these unusual translational events. ...
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... display is a high-throughput molecular technique that has been used successfully to select targets for any given ligand. Targets can be easily displayed on the surface of the phage virion by coupling the foreign DNA to a gene encoding a phage coat protein. After infection of the host, phage protein components are produced by the protein translation machinery of the infected bacterial cell and the foreign DNA will be displayed as a fusion product to one of the phage coat proteins [1]. Because of the physical link between genotype and phenotype, filamentous phage displaying a relevant polypeptide will be retained during affinity selections on candidate binding ligands followed by identification of the target [2]. The most commonly used phage coat proteins for fusion are minor coat protein 3 (p3) and major coat protein 8 (p8) [3,4]. When using these coat proteins, N-terminal fusion of the target is mandatory for successful phage propagation. For the display of cDNA libraries, N-terminal fusion is not possible due to inherent stop codons present in the cDNA fragments, as represented in Figure 1. However, the free carboxyl terminus of minor coat protein 6 (p6) allows successful fusion of the cDNA without interfering with phage propagation [5]. Using the pSP6 phagemid vector which was specifically designed to enable C-terminal fusion of targets to p6, we and others have already successfully identified a variety of targets [5-11]. In our previous studies using C-terminal fusion of cDNA to p6, we observed that during affinity selections a small percentage of phages containing a stop codon immediately after gene VI (gVI) became enriched. This stop codon resulted from an out of frame insertion of the cDNA into the phagemid vector thereby preventing the display of the corresponding protein. Although we initially thought that the isolation of these clones could be the result of aspecific binding, the enrichment of identical cDNA sequences containing this stop codon suggested specific interactions of a displayed target with a ligand. This display could occur when unusual translational recoding such as frameshifting or ribosome hops take ...
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... is well established within the field of phage display and bacterial expression systems [12-16]. Because previous studies were based on N-terminal fusion to p3 or p8, frameshifting had to occur in order to express the p3 and p8 coat proteins that enable phage propagation. However, C-terminal fusion to p6 does not require expression of the inserted cDNA for successful phage propagation as represented in figure 1 [5,6]. Therefore, other methods must be used to detect the possible occurrence of these unusual translational events. In the work described here, a phage clone UH-FS was chosen for further study of frameshifting in the p6 display system. UH-FS was previously isolated after affinity selecting a multiple sclerosis (MS) cDNA display library against antibodies present in MS sera [10]. Although the inserted cDNA sequence of the phage clone encodes part of the Apolipoprotein E protein (ApoE), out of frame insertion of the cDNA sequence into the pSP6 phagemid vector resulted in an early stop codon thereby preventing the display of the protein. Fusion of an E-tag to the ApoE cDNA and subcloning of the fragment in 3 reading frames in the pSP6 vector resulted in the expression of the ApoE - E-tag construct in all reading frames. Measuring expression of both the ApoE polypeptide and the E-tag via ELISA revealed an increased expression both in the correct reading frame but also in the +1 reading frame, indicating the occurrence of frameshifting in the p6 display system. In addition we could demonstrate an increased antibody reactivity towards the correctly displayed ApoE polypeptide in the plasma of the MS patients used for the selection rounds, which strongly indicates the specific enrichment of UH-FS. An overview of the study is represented in figure 2. The inserted cDNA sequence of UH-FS encoded part of the signal sequence in addition to the first 130 amino acids (AA) of the ApoE protein. However, due to out of frame insertion of the ApoE gene in the pSP6 phagemid vector, a stop codon was observed immediately after gVI that prevented the display of the corresponding ApoE polypeptide. In order to detect whether this clone could be subjected to frameshifting, an E-tag was cloned in frame at the 3’-end of the ApoE cDNA as depicted in Figure 2A in order to obtain simultaneous expression of the ApoE polypeptide and the E-tag. This ApoE - E-tag construct was then subcloned into the pSP6 vector in 3 different reading frames (Figure 2E). This resulted in the insertion of the ApoE - E-tag in the 0 frame (designated UH-FSE 0 , Figure 3A) which represents the correct reading frame for the ApoE polypeptide to be expressed, the − 1 frame (designated UH-FSE − 1 , Figure 3B) where a stop codon prevents the display of a peptide and the +1 frame (designated UH-FSE +1 , Figure 3C) in which an artificial peptide of 17 AA is displayed at the surface of the phage. As a control for E-tag expression, the E-tag alone was inserted in the pSP6 vector in the 0 frame (designated UH-PC) and in the − 1 frame (designated UH-NC) resulting in respectively high and no E-tag expression (Figure ...
Citations
... Phage display technology has been extensively used to identify specific binding peptides for many biological molecules including toxins, bacteria, organs, and tumor-associated antigens [79][80][81][82]. Although all five coat proteins have been used to display foreign proteins, the most common approach is to fuse foreign sequences to the Nterminus of p3 and p8 coat proteins [83][84][85]. ...
Viruses have recently emerged as promising nanomaterials for biotechnological applications. One of the most important applications of viruses is phage display, which has already been employed to identify a broad range of potential therapeutic peptides and antibodies, as well as other biotechnologically relevant polypeptides (including protease inhibitors, minimizing proteins, and cell/organ targeting peptides). Additionally, their high stability, easily modifiable surface, and enormous diversity in shape and size, distinguish viruses from synthetic nanocarriers used for drug delivery. Indeed, several plant and bacterial viruses (e.g., phages) have been investigated and applied as drug carriers. The ability to remove the genetic material within the capsids of some plant viruses and phages produces empty viral-like particles that are replication-deficient and can be loaded with therapeutic agents. This review summarizes the current applications of plant viruses and phages in drug discovery and as drug delivery systems and includes a discussion of the present status of virus-based materials in clinical research, alongside the observed challenges and opportunities.
... A surprise for some early practitioners of phage display was that some active encoding sequences had frame disruptions. For instance, biopanning of a random peptide library on a filamentous phage for sequences that would bind to a growth hormone binding protein yielded sequences whose decoding was deduced to undergo substantial levels of either +1 or −1 frameshifting (645), and corresponding results have been seen in other phage display studies (646,647). Recent systematic exploration of shift-prone sequences in E. coli (195), and searches in other bacteria (198) is relevant to the anticipation, and understanding of, such results. ...
Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.
... Peptides and proteins are commonly displayed as N-terminal fusions to coat proteins p3 or p8 ( Fig. 1A; such vectors 8 are dubbed type 3 or 8, respectively), however, all structural proteins can be used as display anchors. Display on C-termini has also been reported for p3, 17 p8, 18,19 p6, [20][21][22][23] and p9 24 , the latter with only limited success. While presentation of short peptides is readily tolerated, large proteins interfere with virion infectivity or prevent capsid assembly due to steric hindrance. ...
... On the other hand, due to the size of phage probes their use in immunocytochemistry is not anticipated as there are unlikely to produce the resolution achieved by much smaller antibodies. 23 The presence of functional groups of different reactivity on virion capsid allows selective attachment of multiple synthetic moieties to phage scaffold. Li et al. 43 exploited noncompeting amine and tyrosine conjugation to couple folic acid and fluorescent dyes, respectively, to filamentous phage. ...
Bacteriophages have been exploited as cloning vectors and display vehicles for decades owing to their genetic and structural simplicity. In bipartite display setting, phage takes on the role of a handle to which two modules are attached, each endowing it with specific functionality, much like the Swiss army knife. This concept offers unprecedented potential for phage applications in nanobiotechnology. Here, we compare common phage display platforms and discuss approaches to simultaneously append two or more different (poly)peptides or synthetic compounds to phage coat using genetic fusions, chemical or enzymatic conjugations, and in vitro non-covalent decoration techniques. We also review current reports on design of phage frameworks to link multiple effectors, and their use in diverse scientific disciplines. Bipartite phage display had left its mark in development of biosensors, vaccines, and targeted delivery vehicles. Furthermore, multi-functionalized phages have been utilized to template assembly of inorganic materials and protein complexes, showing promise as scaffolds in material sciences and structural biology, respectively.
... As the immunoglobulin fold of antibodies generally requires the oxidizing environment of the periplasm to fold, pVI has never been an attractive display route, as only fusion to the pVI C-terminal end is possible and this never leaves the reducing cytosol of E. coli prior to virion incorporation [34,84,85]. During early studies it was also postulated that pVII and pIX would be unsuitable as display scaf- folds due to small size and the assumption that they were com- pletely embedded in the virion coat [84,86]. ...
Phage display technology has evolved to become an extremely versatile and powerful platform for protein engineering. The robustness of the phage particle, its ease of handling and its ability to tolerate a range of different capsid fusions are key features that explain the dominance of phage display in combinatorial engineering. Implementation of new technology is likely to ensure the continuation of its success, but has also revealed important short comings inherent to current phage display systems. This is in particular related to the biology of the two most popular display capsids, namely pIII and pVIII. Recent findings using two alternative capsids, pVII and pIX, located to the phage tip opposite that of pIII, suggest how they may be exploited to alleviate or circumvent many of these short comings. This review addresses important aspects of the current phage display standard and then discusses the use of pVII and pIX. These may both complement current systems and be used as alternative scaffolds for display and selection to further improve phage display as the ultimate combinatorial engineering platform.
The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the –1 frame, occur at specific evolutionarily optimized “slippery” sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry–based method called “translational tiling proteomics” for interrogating the full set of possible –1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical “slippery” sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed.
The phage display technology (PDT) was unique in genetic engineering and recombinant expression. The phage display systems (PDS) were platforms (kits) composed of genetic modified phages, helper phages, and host bacteria. This review concisely summarized the development of four types of PDS, based on M13, λ, T4, and T7 phages, in terms of phage molecular genetics and genetic (gene or genome) engineering. We addressed on the key components and their genetic (genomic) engineering for modifications, the technical features of different anchors, and the development progress and selection reference of those different kits.
