Phage display technology is enabled by genetic fusion of a foreign protein domain to a phage coat protein, without interfering with the phage's ability to replicate by infecting bacterial host cells. The displayed domain is exposed on the phage particle (virion) surface, where it can interact with molecules or other substances in the surrounding medium; in this regard, it acts like a normal protein. However, it possesses a superpower that is unavailable to ordinary proteins: It is easily replicated in great abundance because it is attached to a replicating virion whose genome includes its coding sequence. The main way this technology is exploited is construction of huge phage display “libraries,” comprising billions of phage clones, each displaying a different protein domain, and each represented by thousands, millions, or billions of genetically identical virions—all mixed together in a single vessel. Surface display allows exceedingly rare virions whose displayed protein domains happen to bind a user-defined molecule or other substance—generically called the “selector”—to be isolated from such libraries by an affinity selection process. The yield of selector-binding virions is much too low to be of practical use, but their number is readily increased by many orders of magnitude by propagating the virions in host bacteria in culture. This overview is a critical review of recent developments of this technology. It does not review the entire arena of contemporary phage display; there is special emphasis on phage display's most prominent application, phage antibodies, in which the displayed domain is an antibody domain, and the selector is an antigen of interest.

The most common application of phage-display technology is the discovery of peptides or proteins that specifically bind some molecule or other substance of interest—for example, antibodies that specifically bind an antigen. The discovery process starts with a library encompassing a very large array of proteins or peptides with a great diversity of binding specificities—for example, single-chain antibodies with a great diversity of antigen-binding sites. Each member of the array is displayed on the surface of hundreds to billions of identical virus particles (virions) belonging to a single-phage clone; the library as a whole comprises millions to billions of such clones, all mixed together in a single vessel. Affinity selection is the process by which a molecule or substance of interest—generically called the selector—is used to select very rare clones in the library displaying proteins or peptides that happen to bind the selector with high affinity and selectivity. Here, I explain general principles guiding a successful affinity-selection project—principles grounded in phage biology, kinetics of reversible binding, technological advances, and the practical experience of thousands of investigators around the globe.

Scientific discoveries and technological advances emerge from global communities of inves- tigators sharing their ideas and material resources with one another. Individual investigators or groups make important contributions, to be sure, but these advances are incremental; only when ideas and resources are shared with the community at large, so they can modify and combine with other ideas and resources, does the community become fully creative and innovative. While the "intellectual property" that an individual can legitimately lay claim to is thus very modest, it is the source of a supremely valuable cultural reward: the esteem of the creative communities to which the investigator belongs. When governments attempt to foster innovation by the promise of government-enforced patent monopolies, rather than by direct public spending, scientific communities are disrupted, creativity is impaired, the price of goods and services is greatly elevated, wealth inequality increases, and perverse incentives can distort the marketplace. These shortcomings are especially troubling in the case of pharmaceuticals and other essential sectors, where the products are a public good — not just commodities like TVs or new running shoes — and where effective innovation is a matter of basic economic and social justice — not just commercial prosperity.

Scientific discoveries and technological advances emerge from global communities of investigators sharing their ideas and material resources with one another. Individual investigators or groups make important contributions, to be sure, but these advances are incremental; only when ideas and resources are shared with the community at large, so they can modify and combine with other ideas and resources, does the community become fully creative and innovative. While the "intellectual property" that an individual can legitimately lay claim to is thus very modest, it is the source of a supremely valuable cultural reward: the esteem of the creative communities to which the investigator belongs. When governments attempt to foster innovation by the promise of government-enforced patent monopolies, rather than by direct public spending, scientific communities are disrupted, creativity is impaired, the price of goods and services is greatly elevated, wealth inequality increases, and perverse incentives can distort the marketplace. These shortcomings are especially troubling in the case of pharmaceuticals and other essential sectors, where the products are a public good - not just commodities like TVs or new running shoes - and where effective innovation is a matter of basic economic and social justice - not just commercial prosperity.

Spielen mit der Evolution: Die Anfänge der Entwicklung des Phagen-Display schildert George P. Smith aus persönlicher Sicht in seinem Nobelaufsatz. Die Erfindung dieser Technik ist ein Paradebeispiel dafür, wie wissenschaftlicher Fortschritt allmählich in kleinen Schritten innerhalb sich überlappender globaler wissenschaftlicher Gemeinschaften abläuft.

ABSTRACTTo provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogenAgrobacterium tumefaciens. Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains ofA. tumefaciens. The coinoculation ofA. tumefacienswith phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages.In silicoattempts to find a canonical lysis cassette were unsuccessful; however, we found a putativephagepeptidoglycanhydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression ofpphin the absence of additional phage genes causes a block in cell division and subsequent lysis ofA. tumefacienscells. When the presumed active site of theN-acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic.IMPORTANCEThe characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such asAgrobacterium tumefaciens, may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The specificity of bacteriophage treatment for the host is an asset in complex communities, such as in orchards where it would be detrimental to harm the symbiotic bacteria in the environment. Second, bacteriophages are potential sources of enzymes that efficiently lyse bacterial cells. These phage proteins may have a broad specificity, but since proteins do not replicate as phages do, their effect is highly localized, providing an alternative to traditional antibiotic treatments. Thus, studies of lytic bacteriophages that infectA. tumefaciensmay provide insights for designing preventative strategies against bacterial pathogens.

Reversible binding between biomolecules—for example, between a cell-surface receptor such as the insulin receptor and its corresponding natural ligand such as insulin—is central to innumerable physiological transactions. Binding of the dye HABA to egg-white avidin is a simple, reliable, and colorful laboratory model for introducing beginning biology students to the principles underlying reversible binding. They can probe the reaction quantitatively with a spectrophotometer, and model it mathematically using only high-school algebra and a spreadsheet program such as Microsoft Excel.

This paper calculates probability distributions modeling the Luria-Delbrück experiment. We show that by thinking purely in terms of generating functions, and using a 'backwards in time' paradigm, that formulas describing various situations can be easily obtained. This includes a generating function for Haldane's probability distribution due to Ycart. We apply our formulas to both simulated and real data created by looking at yeast cells acquiring an immunization to the antibiotic canavanine. This paper is somewhat incomplete, having been last significantly modified in March 29, 2014. However the first author feels that this paper has some worthwhile ideas, and so is going to make this paper publicly available.

Microbial cultures swiftly adapt to lethal agents such as antibiotics or viruses by acquiring resistance mutations. Does this remarkable adaptability require a Lamarckian explanation, whereby the agent specifically directs resistance mutations? Soon after the question arose, Luria and Delbruck devised a clever experiment, the fluctuation test, that answered this question in the negative: microbial adaptation, they showed, is entirely consistent with a Darwinian explanation. Their 1943 article is a classic of biology literature, with practical and theoretical implications that continue to expand today. Implementing an updated fluctuation test in a college teaching lab provides a simple experimental setting in which beginning students learn to apply basic principles of evolutionary biology and scientific reasoning, while gaining hands-on experience in core technical advances of contemporary life science.

Construction of molecular chimeras from different sources has been routine in molecular biology since gene splicing began in the middle of 1970s. For a detailed discussion of the genetics and biochemistry of molecular cloning systems, refer to the comprehensive survey of vectors edited by and Denhardt In 1985, recombinant DNA techniques were used to fashion a new type of chimera that underlies today’s phage display technology (2). To create one of these chimeras, a foreign coding sequence is spliced in-frame into a phage coat protein gene, so that the ‘‘guest’’ peptide encoded by that sequence is fused to a coat protein and thereby displayed on

Purpose The goal of this study was to improve the pharmacokinetic properties and specificity of an ERBB2-targeted peptide for SPECT imaging. Procedures Bacteriophages (phages) displaying the ERBB2 targeting sequence, KCCYSL, flanked by additional random amino acids were used for in vivo selections in mice-bearing ERBB2-expressing MDA-MB-435 human breast xenografts. Phage-displayed peptides were evaluated for ERBB2 and cancer cell binding affinity and specificity in vitro , and one peptide was radiolabeled with 111 In-DOTA and biodistribution and SPECT imaging properties were compared to the first generation peptide, 111 In-DOTA-KCCYSL. Results In vivo phage display selected two peptides, 1-D03 (MEGPSKCCYSLALSH) and 3-G03 (SGTKSKCCYSLRRSS), with higher breast carcinoma cell specificity and similar ErbB2 affinity (236 and 289 nM, respectively) to the first generation peptide. The corresponding radiolabeled probes bound with higher affinity to target cancer cells than 111 In-DOTA-KCCYSL; however, only 111 In-DOTA-1-D03 demonstrated higher specificity for MDA-MB-435 cells. Biodistribution analysis demonstrated that although 111 In-DOTA-1-D03 had slightly reduced tumor uptake (0.661 % ID/g) in comparison to 111 In-DOTA-KCCYSL (0.78 %/ID/g), its dramatic improvement in blood clearance led to a significantly higher tumor/blood ratio (6.02:1). Non-specific uptake was also reduced in most organs including heart, lung, muscle, bone, and kidneys. SPECT imaging revealed tumor-specific uptake of 111 In-DOTA-1-D03, which was confirmed by blocking with unlabeled 1-D03 peptide. Conclusions This is the first evidence that SPECT imaging peptides with improved tumor specificity and pharmacokinetics can be obtained by in vivo phage display affinity maturation. The combination of ERBB2-specific binding, rapid clearance, and tumor specificity may make 1-D03 a viable candidate for clinical imaging studies.

Clinical use of most radiolabeled targeting agents has been limited because of the uptake and retention in kidney and/or liver. We hypothesized that bacteriophage (phage) display could be exploited to select for peptide sequences with fast clearance and low kidney uptake with the added ability to redirect phage clearance away from the reticuloendothelial system towards the kidney possessing rapid kidney clearance.

This chapter will review the physical and biological properties of filamentous phage that have made them a flexible platform for development of new nanoparticles. The long, thin virion is extraordinarily robust, being tolerant of harsh physical conditions, extensive chemical modification, and genetic fusion of foreign “guest” peptides and proteins to its exposed coat proteins. As much as twenty percent of its outer surface can be modified without impairing phage assembly or infectivity. Virions can be reliably produced in great abundance and purified to a high degree of purity by simple, scalable processes. Most importantly, their ability to replicate has opened up invention strategies that depend on selecting particles with desired properties from vast populations with random variations, rather than on rational design. Growing knowledge of the unusual infection cycle, along with more than three decades of experience in manipulating filamentous phages for biotechnological ends, have resulted in an extensive “toolkit” of useful, broadly applicable techniques for enhancing a new initiative's chance of success.