Yeast cell-surface display—applications of molecular display

In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.     

Do neocortical pyramidal neurons display stochastic resonance?

Neocortical pyramidal neurons in vivo are subject to an intense synaptic background activity that has a significant impact on various electrophysiological properties and dendritic integration. Using detailed biophysical models of a morphologically reconstructed neocortical pyramidal neuron, in which synaptic background activity was simulated according to recent measurements in cat parietal cortex in vivo, we show that the responsiveness of the cell to additional periodic subthreshold stimuli can be significantly enhanced through mechanisms similar to stochastic resonance. We compare several paradigms leading to stochastic resonance-like behavior, such as varying the strength or the correlation in the background activity. A new type of resonance-like behavior was obtained when the correlation was varied, in which case the responsiveness is sensitive to the statistics rather than the strength of the noise. We suggest that this type of resonance may be relevant to information processing in the cerebral cortex.     

Phage display: a useful tool for malaria research?

Defining the molecular intricacies of malaria pathogenesis is a vital area of medical and scientific research. Sophisticated methods have been developed to identify and characterise host-parasite interactions that are important in infection. Phage display involves the combinatorial display of proteins or peptides on the surface of bacteriophage. The technology provides an invaluable tool for screening diverse libraries for polypeptides that have a high affinity for a given target. Phage display in malaria research has proven successful, not only in mapping the protein-protein interactions that are important in Plasmodium biology, but also in the identification of molecules that might be exploited in the design of therapeutic agents or vaccines.     

Does titanium in ionic form display a tissue-specific distribution?

Most studies have focused on the biodistribution of titanium(IV) oxide as nanoparticles or crystals in organism. But several reports suggested that titanium is released from implant in ionic form. Therefore, gaining insight into toxicokinetics of Ti ions will give valuable information, which may be useful when assessing the health risks of long-term exposure to titanium alloy implants in patients. A micro synchrotron radiation-induced X-ray fluorescence (µ-SRXRF) was utilized to investigate the titanium distribution in the liver, spleen and kidneys of rats following single intravenous or 30-days oral administration of metal (6 mg Ti/b.w.) in ionic form. Titanium was mainly retained in kidneys after both intravenous and oral dosing, and also its compartmentalization in this organ was observed. Titanium in the liver was non-uniformly distributed—metal accumulated in single aggregates, and some of them were also enriched in calcium. Correlation analysis showed that metal did not displace essential elements, and in liver titanium strongly correlated with calcium. Two-dimensional maps of Ti distribution show that the location of the element is characteristic for the route of administration and time of exposure. We demonstrated that µ-SRXRF can provide information on the distribution of titanium in internal structures of whole organs, which helps in enhancing our understanding of the mechanism of ionic titanium accumulation in the body. This is significant due to the popularity of titanium implants and the potential release of metal ions from them to the organism.     

Mapping protein–ligand interactions using whole genome phage display libraries

The function of many genes cannot be deduced from sequence similarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. The pJuFo phage display system, based on the interaction between the leucine zippers Jun and Fos, has been modified and used to create a genomic phage display library from Escherichia coli MG1655. The system has been tested by using the library to map the dominant binding epitopes for an anti-RecA protein polyclonal antibody sera. This methodology provides a general biochemical approach to functional analysis of protein–ligand interactions on a genome-wide basis.     

Shotgun phage display — Selection for bacterial receptins or other exported proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins. Published: May 1, 2003     

Trees and/or networks to display intraspecific DNA sequence variation?

Phylogenetic trees and networks are both used in the scientific literature to display DNA sequence variation at the intraspecific level. Should we rather use trees or networks? I argue that the process of inferring the most parsimonious genealogical relationships among a set of DNA sequences should be dissociated from the problem of displaying this information in a graph. A network graph is probably more appropriate than a strict consensus tree if many alternative, equally most parsimonious, genealogies are to be included. Within the maximum parsimony framework, current phylogenetic inference and network‐building algorithms are both unable to guarantee the finding of all most parsimonious (MP) connections. In fact, each approach can find MP connections that the other does not. Although it should be possible to improve at least the maximum parsimony approach, current implementations of these algorithms are such that it is advisable to use both approaches to increase the probability of finding all possible MP connections among a set of DNA sequences.     

Engineering human interferon α1c/86D with phage display technology

Human interferon-α1c/86D (IFNα1c/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopes on the IFN-α1b, indicating that residues 30, 33, 34, (AB-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-hased panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4—16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN α1c/86D variants with increased specific activity might be obta     

Analysis of waving and sound‐production display in the ghost crab,Ocypode stimpsoni

The ghost crab Ocypode stimpsoni displays waving and sound production. Sounds are produced by thumping the sand substratum with the major cheliped, and two types of sounds can be discriminated; one with a low frequency of about 12 Hz, called rapping, and another with a higher frequency (about double), called quivering. In our observations, a sequence of waving and sound emission would sometimes terminate abruptly, or appear as independent components but the component order never changed. The most frequently observed patterns were “waving with rapping and quivering”;, “waving with quivering”; and “quivering only”; quivering sounds being involved in more than 80% displays. Quivering sometimes occurred immediately after crabs emerged from the burrow, or when they returned to the entrance after discarding an excavated sand mass. The occurrence frequency of waving and sounds, the wave amplitude, and the frequency of the sound increased when other crabs approached.     

Selection of bisphenol A – single-chain antibodies from a non-immunized mouse library by ribosome display

Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KD_S) of one clone was 1.76 μM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies.     

Agonistic display or courtship behavior? A review of contests over mating opportunity in butterflies

Male butterflies compete over mating opportunities. Two types of contest behavior are reported. Males of various butterfly species compete over a mating territory via aerial interactions until one of the two contestants retreats. Males of other butterfly species fly around larval food plants to find receptive females. Males of some species among the latter type can find a conspecific pupa, and they gather around it without expelling their rivals. Scramble competition over mating occurs when a female emerges from the pupa. Many studies have been performed on territorial species, and their contest resolution has often been understood from the point of view of contest models based on game theory. However, these models cannot explain why these butterflies perform contest displays despite the fact that they do not have the ability to attack their opponent. A recent study based on Lloyd Morgan’s Canon showed that territorial contests of male butterflies are better understood as erroneous courtship between sexually active males. In this paper, I review research on contests over mating opportunity in butterflies, and show that the erroneous courtship framework can explain not only territorial contests of butterflies but also why males do not determine the owner of a conspecific pupa.     

Selectable in-vivo recombination to increase antibody library size — an improved phage display vector system

Phage display technology permits the display of libraries of random combinations of light (LC) and heavy chain (HC) antibody genes. Maximizing the size of these libraries would enable the isolation of antibodies with high affinity and specificity. In this study, the loxP/Cre system of in-vivo recombination has been employed to construct an improved vector system for the display of antibodies. In this system, the chloramphenicol acetyl transferase (CAT) gene is linked to a HC library in a donor plasmid, pUX. This CAT gene is `silent' before recombination but active after recombination. A second acceptor phagemid, pMOX, is used for cloning the LC repertoire. Following infection with a Cre producing phage, pMOX accepts the CAT/HC library from pUX via site-specific recombination at the loxP sites. Recombinants can then be selected via chloramphenicol resistance. Using this vector system, we have generated libraries of 4×10⁹ recombinants. Restriction analysis and Fab expression confirmed that 100% of the colonies in the library were recombinants. This system provides a stable selectable mechanism for the generation of large libraries and avoids the isolation of non-recombinants encountered with earlier in-vivo recombination systems.     

That’s hot: golden spiny mice display torpor even at high ambient temperatures

Golden spiny mice (Acomys russatus) living in the Judean desert are exposed to extended periods of food and water shortage. We investigated their thermal and metabolic response to three weeks of 50 % food reduction at ambient temperatures of 23, 27, 32 and 35 °C by long term records of metabolic rate and body temperature in the laboratory. At all ambient temperatures, A. russatus responded to starvation by a reduction of daily energy expenditure. At 32 and 35 °C, this metabolic adjustment fully compensated the reduced food availability and they maintained their energy balance at a slightly reduced body mass. At lower ambient temperatures, they could not fully compensate for the reduced food availability and kept a negative energy balance. The reduction of daily energy expenditure was largely achieved by the occurrence of daily torpor. Torpor even occurred at high ambient temperatures of 32 and 35 °C during which metabolic depression was not associated with a marked decrease of body temperature. The results show that the occurrence of daily torpor is not necessarily linked to cold exposure and the development of a pronounced hypothermia, but may even occur as depression of metabolic rate in a hot environment.     

Surface display of N-terminally anchored invasin by Lactobacillus plantarum activates NF-κB in monocytes

The probiotic lactic acid bacterium Lactobacillus plantarum is a potential delivery vehicle for mucosal vaccines because of its generally regarded as safe (GRAS) status and ability to persist at the mucosal surfaces of the human intestine. However, the inherent immunogenicity of vaccine antigens is in many cases insufficient to elicit an efficient immune response, implying that additional adjuvants are needed to enhance the antigen immunogenicity. The goal of the present study was to increase the proinflammatory properties of L. plantarum by expressing a long (D1 to D5 [D1-D5]) and a short (D4-D5) version of the extracellular domain of invasin from the human pathogen Yersinia pseudotuberculosis. To display these proteins on the bacterial surface, four different N-terminal anchoring motifs from L. plantarum were used, comprising two different lipoprotein anchors, a transmembrane signal peptide anchor, and a LysM-type anchor. All these anchors mediated surface display of invasin, and several of the engineered strains were potent activators of NF-κB when interacting with monocytes in cell culture. The most distinct NF-κB responses were obtained with constructs in which the complete invasin extracellular domain was fused to a lipoanchor. The proinflammatory L. plantarum strains constructed here represent promising mucosal delivery vehicles for vaccine antigens.     

Engineering of Escherichia coli β-galactosidase for solvent display of a functional scFv antibody fragment

Protein engineering allows the generation of hybrid polypeptides with functional domains from different origins and therefore exhibiting new biological properties. We have explored several permissive sites in Escherichia coli β-galactosidase to generate functional hybrid enzymes displaying a mouse scFv antibody fragment. When this segment was placed at the amino-terminus of the enzyme, the whole fusion protein was stable, maintained its specific activity and interacted specifically with the target antigen, a main antigenic determinant of foot-and-mouth disease virus. In addition, the antigen-targeted enzyme was enzymatically active when bound to the antigen and therefore useful as a reagent in single-step immunoassays. These results prove the flexibility of E. coli β-galactosidase as a carrier for large-sized functional domains with binding properties and prompt the further exploration of the biotechnological applicability of the scFv enzyme targeting principle for diagnosis or other biomedical applications involving antigen tagging.     

Surface display expression of Bacillus licheniformis lipase in Escherichia coli using Lpp’OmpA chimera

The lipase from Bacillus licheniformis ATCC14580 was displayed on the cell surface of Escherichia coli using Lpp’OmpA as the anchoring protein. The expressed Lpp’OmpA-lipase fusion protein has a molecular weight of approximately 35 kDa, which was confirmed by SDS-PAGE and western blot analysis. The Lpp’OmpA-lipase fusion protein was located on the cell surface, as determined by immunofluorescence confocal microscopy and flow cytometry. The enzyme activity of the surface-displayed lipase showed clear halo around the colony. The cell surface-displayed lipase showed the highest activity of 248.12 ± 9.42 U/g (lyophilized cell) at the optimal temperature of 37°C and pH 8.0. The enzyme exhibited the highest activity toward the substrate p-nitrophenyl caprylate (C8). These results suggest that E. coli, which displayed the lipase on its surface, could be used as a whole cell biocatalyst.