Bacterial display in combinatorial protein engineering

Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.     

Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies

The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.     

DeltaPhage--a novel helper phage for high-valence pIX phagemid display

Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.     

Development of yeast molecular display systems focused on therapeutic proteins, enzymes, and foods: functional analysis of proteins and its application to bioconversion

Molecular display systems using yeast have been developed for industrial, medical, pharmaceutical, and biological studies. Although several host cells are available to construct a molecular display system, the yeast Saccharomyces cerevisiae is a well-established and convenient organism in eukaryotes. A wide variety of prokaryotic and eukaryotic proteins have been displayed on yeast cell surfaces. In addition, functional analyses and applications to bioconversion have been performed on the cell surface, and cells are conveniently engineered by molecular display systems. In this review, we focus on the yeast molecular display system with regard to therapeutic proteins, several enzymes, and food ingredients. In addition, recent patents on molecular display using yeast cell for production of those compounds, screening technology and related techniques are introduced. Development of devices for functional analysis of created and modified proteins in the yeast display system is also described.     

Courtship behaviour in a lekking species: individual variations and settlement tactics in male little bustard

We analysed the display behaviour of male little bustard Tetrax tetrax to identify displays that are used in the context of male-male competition and those that are used for attracting females. Courtship was the main activity of males during the breeding season. Calling activity occurred throughout the day, and leks were attended for more than 4 months. Male sexual displays included snort call, wing-flash, and jump display. Snort call was performed throughout the day and mainly involved male-male interactions. In contrast, the wing-flash display was given only at twilight, and was performed most commonly when a female was present, supporting an inter-sexual function for this display. The jump display was performed only in the presence of female at anytime of the day. Analysis of individual variations in display behaviour revealed that intra-individual variation was low compared to inter-individual variation, especially for the jump display. It is, therefore, possible that display rates provide information on male quality. Four male settlement patterns could be defined, singles, paired, lekking and satellite lekking, but only wing-flash display and stamped snort call differed among those categories. We suggest that satellite males are attempting to benefit from proximity to higher status males, in accordance with the hotshot hypothesis of lek evolution.     

Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles

Filamentous phages are now the most widely used vehicles for phage display and provide efficient means for epitope identification. However, the peptides they display are not very immunogenic because they normally fail to present foreign epitopes at the very high densities required for efficient B-cell activation. Meanwhile, systems based on virus-like particles (VLPs) permit the engineered high-density display of specific epitopes but are incapable of peptide library display and affinity selection. We developed a new peptide display platform based on VLPs of the RNA bacteriophage MS2. It combines the high immunogenicity of MS2 VLPs with the affinity selection capabilities of other phage display systems. Here, we describe plasmid vectors that facilitate the construction of high-complexity random sequence peptide libraries on MS2 VLPs and that allow control of the stringency of affinity selection through the manipulation of display valency. We used the system to identify epitopes for several previously characterized monoclonal antibody targets and showed that the VLPs thus obtained elicit antibodies in mice whose activities mimic those of the selecting antibodies.     

Sexual selection and the mating system of Argusianus argus (Aves: Phasianidae)

A study of the Great Argus pheasant Argusianus argus is described. Cleared display sites are owned each by a single male which calls to attract females, while other adult and subadult males maintain no such sites. Evidence is presented to show that vocal differences between males at display sites are slight and unstable, and are therefore unlikely to be used by females in mate choice. No evidence was obtained to suggest that females wander from display site to site and mate with the best male chosen by visual comparison of subtle plumage variation, although the classical assumption has been that such choice accounts for the evolution of the males' extravagant plumage. Females may by chance meet males without display sites at random in the forest, but the displays of such males are probably insufficiently stimulating to induce the female to copulate. Choice between males with or without display sites, on gross plumage and display differences, provides an alternative explanation for the evolution of the males' plumage.     

Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage

Background

Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.

Methodology/Principal Findings

Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.

Conclusions/Significance

Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.     

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.     

体外展示技术及其在抗体工程中的应用

体外展示技术包括核糖体展示技术、mRNA展示技术、DNA展示技术,是在无细胞蛋白质表达体系内将基因型和表型通过一定的方法连接在一起,体外高通量的筛选多肽和蛋白质的技术。抗体的产生是一个不断选择的过程,利用体外展示技术在体外选择针对某一抗原的抗体分子,并结合基因工程技术对抗体进行改造,以产生高亲和力、高特异性的抗体。体外展示技术的研究和应用已越来越广泛,有望成为下一代的抗体制备技术。     

Survival costs and reproductive benefits of floral display in a sexually dimorphic dioecious shrub, Leucadendron xanthoconus

The evolutionary causes of sexual dimorphism in plants have not been as widely studied as in animals and the importance of sexual selection in causing dimorphism remains controversial. Sexual selection is most obvious when it favours the evolution of a trait which enhances mating success at the expense of decreased viability. We studied the relationship between floral display (number of inflorescences), pollinator attraction and plant survival in a dioecious shrub, Leucadendron xanthoconus. Pollinator attraction, measured as the number of insect pollinators, increased linearly with floral display in males. However, males with extravagant displays had a higher probability of dying. Our data suggest that male plants are undergoing selection on floral display for increased mating success counterbalanced by selection against plants with extravagant displays. Seed set in females did not increase with floral display, except at very low inflorescence numbers. Nor was female survival correlated with floral display. Because inflorescences are terminal in the species, selection for more inflorescences in males causes increased ramification, thinner terminal branches and smaller leaves. Thus vegetative dimorphism in this species appears to be caused by selection for extravagant floral display in males, but not females. Limits to dimorphism are imposed by survival costs of elaborate display.     

Decorating microbes: surface display of proteins on Escherichia coli

Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. By contrast, display systems based on autotransporter proteins (ATs) and ice nucleation protein (INP) are excellent systems for the display of large and complex proteins, and are therefore of considerable biotechnological relevance. Here, we review recent advances in AT and INP-mediated display and their biotechnological applications. Additionally, we discuss several promising alternative display methods, as well as novel bacterial host organisms.     

In vivo phage display — A discovery tool in molecular biomedicine

In vivo phage display is a high-throughput method for identifying target ligands specific for different vascular beds. Targeting is possible due to the heterogeneous expression of receptors and other antigens in a particular vascular bed. Such expression is additionally influenced by the physiological or pathological status of the vasculature. In vivo phage display represents a technique that is usable in both, vascular mapping and targeted drug development. In this review, several important methodological aspects of in vivo phage display experiments are discussed. These include choosing an appropriate phage library, an appropriate animal model and the route of phage library administration. In addition, peptides or antibodies identified by in vivo phage display homing to specific types of vascular beds, including the altered vasculature present in several types of diseases are summarized. Still, confirmation in independent experiments and reproduction of identified sequences are needed for enhancing the clinical applicability of in vivo phage display research.     

Evolution of Anoline Lizard Display Behavior

Based on my conceptual framework of anoline display behavior,I am suggesting the following evolutionary trends. Lateral presentationduring display was probably promoted by monocular vision. Alongwith lateral presentation, postures evolved to increase lateraloutline. These postures which magnified body size were probablyof selective advantage within aggressive social contexts sincelarger animals tend to dominate smaller ones through bluff.Body movement evolved along with lateral orientation and size-enhancingpostures. These movements would be most effective if they complementedlateral orientation. Effectors available for such movementswere primarily pre-adapted for vertical motion. The patternsof movement generated were probably simple oscillatory bobbingmovements by the head which were weakly stereotyped, interspecificallysimilar, appearing in many contexts, and having a weakly definedinformation content. Events having selective advantage for speciesrecognition promoted stereotypy of bobbing behavior into species-uniquedisplays; each species had its unique signature display whichserved in a manifold communicatory capacity. The signature displayappeared in assertion, courtship, and challenge contexts. Itsinformation content varied depending upon context and recipientof the display (e.g., male or female). Besides the stereotypedaspects of the display, certain features remained variable withpotential information significance. Core variability (see text)promotes individual recognition and may be the origin of newunique display patterns as sibling species emerge. Display modifiers(see text) are variable display features shared by members ofa population (many being shared interspecifically) that providea graded appearance to display performance; modifiers can indicatelevel of arousal and facilitate interspecific communication.For some species display repertoire size seems to have evolvedfrom a single display (signature display) to repertoires ofmultiple displays; these subsequent displays are generally restrictedto aggressive interactions.     

Phage display systems and their applications

Screening phage display libraries of proteins and peptides has, for almost two decades, proven to be a powerful technology for selecting polypeptides with desired biological and physicochemical properties from huge molecular libraries. The scope of phage display applications continues to expand. Recent applications and technical improvements driving further developments in the field of phage display are discussed.     

酵母表面展示体系的构建及在纤维素降解中的应用*

纤维素是来源广泛且储量较大的低成本可再生资源,但其结构致密难以利用。目前降解纤维素需要多种纤维素酶协作,而游离纤维素酶成本高、难以重复利用等问题限制了其广泛应用。利用酵母表面展示技术,可以将多个纤维素酶分别与锚定蛋白融合后共展示在细胞表面,从而构建酵母表面展示纤维素酶体系。这一体系可高效降解纤维素,一方面可以充分发挥表面展示的优点,如易回收、稳定性好、操作简单、成本低;另一方面可以将纤维素有效地降解为葡萄糖,并具有代谢产生物乙醇的潜力。阐述了酵母表面展示体系的构建原则,总结了影响展示体系效率的因素,介绍了这一技术在降解纤维素中的应用,为构建高效酵母表面展示纤维素酶体系及其他多酶体系提供参考。     

Body Condition in Male Betta splendens Does Not Predict Their Ability to Perform Opercular Displays Under Hypoxic Conditions

Many fish, including the fighting fish Betta splendens , perform a display in which the opercula are extended away from the head and gills. Previous work has shown that opercular display rates by male B. splendens decrease under conditions of reduced dissolved oxygen (hypoxia). We tested the hypothesis that the ability to maintain opercular display rates under hypoxic conditions is related to body condition in male B. splendens . We also tested the hypothesis that females would show a greater preference for males performing this display under hypoxic conditions, when the display should be a more reliable indicator of male phenotypic quality. We found no evidence to support either hypothesis. Male opercular display rate in hypoxic conditions was unrelated to natural or experimentally induced variation in body condition. Female B. splendens showed no differential preference for the opercular display, assessed through the use of computer animated male stimuli, in either acute or chronic hypoxia. We conclude that the presence of an air-breathing organ in this species makes the opercular display an unreliable signal of male quality as measured by body condition.     

Display of an Inedible Prop as a Signal of Aggression? Adaptive Significance of Leaf‐Display by the Turquoise‐Browed Motmot,Eumomota superciliosa

The incorporation of an inedible object (prop) into a behavioral display is rare among birds. Only four avian taxa have been reported to display with a prop, and in all studied species, prop use has been found to play a role in mate acquisition. However, little is known about the context and adaptive significance of prop use by the motmots. Both male and female motmots perform a leaf‐display, whereby a leaf is held horizontally in the tip of the bill for an extended period. I collected observational data on leaf‐display by the turquoise‐browed motmots (Eumomota superciliosa) to investigate the potential role of this display as a mate‐choice or agonistic signal. Observations of 61 leaf‐displays by 27 individuals indicate that males and females are similarly likely to perform the leaf‐display, that the leaf‐display is performed throughout the long‐breeding season, and that the display is performed by both paired and unpaired birds. These observations suggest that the leaf‐display does not play a role in mate‐attraction. However, the leaf‐display was often performed in association with a chase or fight with a same‐sex individual, and the display was sometimes performed in conjunction with an agonistic display at nest sites, suggesting that the leaf‐display may function in both sexes as a signal of aggressive intent or as a signal of status.     

Bicistronic DNA display for in vitro selection of Fab fragments

In vitro display methods are superior tools for obtaining monoclonal antibodies. Although totally in vitro display methods, such as ribosome display and mRNA display, have the advantages of larger library sizes and quicker selection procedures compared with phage display, their applications have been limited to single-chain Fvs due to the requirement for linking of the mRNA and the nascent protein on the ribosome. Here we describe a different type of totally in vitro method, DNA display, that is applicable to heterodimeric Fab fragments: in vitro compartmentalization in water-in-oil emulsions allows the linking of an oligomeric protein and its encoding DNA with multiple ORFs. Since previously used emulsions impaired the synthesis of functional Fab fragments, we modified conditions for preparing emulsions, and identified conditions under which it was possible to enrich Fab fragments 10⁶-fold per three rounds of affinity selection. Furthermore, we confirmed that genes encoding stable Fab fragments could be selected from a Fab fragment library with a randomized hydrophobic core in the constant region by applying heat treatment as a selection pressure. Since this method has all advantages of both phage display and totally in vitro display, it represents a new option for many applications using display methods.     

Cichlid spawning structures – bowers or nests?

Males of mouthbrooding cichlids build sand-castle or sand-scrape structures. These are used as display sites to attract females, eggs are laid and inseminated there and then taken away by the female for brooding elsewhere. It has been suggested that the structure be called a bower because it has the same role as the bowerbird's bower. Thew word bower is restricted in ornithological literature to complex structures which reminded Gould (1840) of garden bowers. Simpler display sites of other bowerbirds and other bird families are called courts. Bowerbirds use separate nests for egg-laying, cichlids do not. Other birds, e.g. many weavers, use nests for display purposes. The cichlid structure is the same as nests used by other non mouthbrooding fishes, but mouthbrooding has freed females from the need to stay in the nest. It is unacceptable to use the word bower for the cichlid structure because it is not a bower as defined in ornithological literature, and it is used for egg laying as well as display. Weaver birds use nests for display in a similar way to cichlids, thus the word nest should be retained for the cichlid sand structure. This revised version was published online in July 2006 with corrections to the Cover Date.