Recent Patents on Biotechnology (v.4, #3)

The patents annotated in this section have been selected from various patent data bases. These recent patents are relevant to the articles published in this journal issue, categorized by different biotechnology methods, processes and techniques involved.

Since the development of genetic engineering in the 1970s, various biotechnological tools have been established. These tools, which use gene manipulation techniques, have been used to manufacture biologically active compounds and pharmaceutics; they have also enabled the production of various important molecules whose production by conventional fermentation technology was difficult. Thus, gene manipulation techniques have enhanced the efficiency of bioindustrial processes. This issue focuses on genetic engineering and combinatorial bioengineering-based molecular display technology that is unlike any other biotechnological method - it includes and#x201C;displayand#x201D;, and#x201C;diversityand#x201D;, and and#x201C;directed selection,and#x201D; (and#x201C;3Dand#x201D;), which are new concepts. Smith reported that a foreign protein could be inserted into filamentous phage protein III, resulting in the display of a fusion protein on the virion surface; this was the first report on molecular display technology. After this, several types of display systems using different organisms were developed. Combinatorial technology was introduced to endow diversity on the displayed protein; with this technology, several new previously undiscovered proteins were identified. For directed selection of useful clones with desired function from among the numerous clones generated, advanced and high-throughput devices such as cell sorters, chamber array systems, and high-speed DNA analyzing systems have played important roles. This special issue consists of 6 review articles on biotechnology, particularly molecular display technologies. The authors featured in this issue are pioneers in their respective fields and present rather interesting topics in molecular display technology. We, as biotechnologists, should be aware of the importance of patents related in our laboratory for experiments. No matter how new the theme, it is bound to be related to other published/patented works. Therefore, these authors not only describe their results but also use and cite other published/patented material. In the first article, Stahl et al. report Staphylococcal surface display in combinatorial protein engineering and its applications. Next, Tsumoto and Ui describe a topic on rational ligand design introducing thermodynamic analysis. In the third article, Ye and Geels describe the design and applications of vaccines by using molecular display technology. In the fourth article, Shibasaki and Ueda focus on a yeast molecular display system for bioconversion. In the fifth article, bacterial magnetic particles and its biotechnological applications are introduced by Matsunaga et al. In the final article of this issue, Kondo et al. describe the applications of molecular display technology for biorefineries. As evident, these are all intriguing, inter-related topics on molecular display technologies. Through this special issue, the guest editors hope that all readers of the journal will gain a deep understanding of the usefulness of molecular display technologies in the development of various tools for bioindustrial and biotechnological research. We would like to express our gratitude to all the authors for their dedication and for generously sharing their recent research results and patents. We would also like to thank all the reviewers of the articles published in this issue. We are grateful to Ms. Tayyaba Zaidi of Bentham Science Publishers for her practical advice and encouragement.

Staphylococcal Surface Display in Combinatorial Protein Engineering and Epitope Mapping of Antibodies by Nina Kronqvist, Magdalena Malm, Johan Rockberg, Barbara Hjelm, Mathias Uhlen, Stefan Stahl, John Lofblom (171-182).
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.

Thermodynamic analysis is an effective tool in screening of lead-compounds for development of potential drug candidates. In most cases, a ligand achieve high affinity and specificity to a target protein by means of both favorable enthalpy and entropy terms, which can be reflected in binding profiles of Isothermal Titration Calorimetry (ITC). A favorable enthalpy change suggests the contribution of noncovalent contacts such as hydrogen bonding and van der Waals interaction between a ligand and its target protein. In general, optimization of binding enthalpy is more difficult than that of entropies in ligand-design; therefore, it is desirable to choose firstly a lead-compound based on its binding enthalpic gain. In this paper, we demonstrate the utility of thermodynamic approach to ligand screening using anti-ciguatoxin antibody 10C9 as a model of a target protein which possesses a large hydrophobic pocket. As a result of this screening, we have identified three compounds that could bind to the antigen-binding pocket of 10C9 with a few kcal/mol of favorable binding enthalpy. Comparison of their structure with the proper antigen ciguatoxin CTX3C revealed that 10C9 rigorously identifies their cyclic structure and a characteristic hydroxyl group. ITC measurement might be useful and powerful for a rational ligand screening and the optimization of the ligand; the enthalpic gain is an effective index for ligand-design studies.

Conventional vaccine production techniques are outdated, leaving the world defenseless to viruses and pathogens. Successful protection necessitates the innovation of strategies that can generate an induced defensive humoral and cellular response with: ease of mass production, nominal side-effects, and controlled design specificity, all while being cost effective. Fortunately, technology exists to facilitate such advances in this billion dollar industry and this review is focused on recent publications and patents which hold promise to revolutionize the fight against pathogenic illnesses.

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.

Bioengineering of Bacterial Magnetic Particles and their Applications in Biotechnology by Tomoko Yoshino, Yoshiaki Maeda, Tadashi Matsunaga (214-225).
Magnetic particles have attracted much attention for their versatile uses in biotechnology, especially in medical applications. The major advantage of magnetic particles is that they can be easily manipulated by magnetic forces. Magnetotactic bacteria synthesize nano-sized biomagnetites, otherwise known as bacterial magnetic particles (BacMPs) that are individually enveloped by a lipid bilayer membrane. The mechanisms of BacMP synthesis have been analyzed by genomic, proteomic, and bioinformatic approaches. Based on those studies in Magnetospirillum magneticum AMB-1, functional nanomaterials have been designed and produced. Through genetic engineering, functional proteins such as enzymes, antibodies, and receptors have been successfully displayed on BacMPs. These functional BacMPs have been utilized in various biosensors and bio-separation processes. Here, recent papers and patents for bioengineering of BacMPs and their applications in biotechnology are reviewed. The elucidation of the mechanism of magnetic particle synthesis has provided a roadmap for the design of novel biomaterials that can play useful roles in multiple disciplinary fields.

Applications of Yeast Cell-Surface Display in Bio-Refinery by Akihiko Kondo, Tsutomu Tanaka, Tomohisa Hasunuma, Chiaki Ogino (226-234).
The dependency on depleting natural resources is a challenge for energy security that can be potentially answered by bioenergy. Bioenergy is derived from starchy and lignocellulosic biomass in the form of bioethanol or from vegetable oils in the form of biodiesel fuel. The acid and enzymatic methods have been developed for the hydrolysis of biomass and for transesterifiaction of plant oils. However, acid hydrolysis results in the production of unnatural compounds which has adverse effects on yeast fermentation. Recent advancements in the yeast cell surface engineering developed strategies to genetically immobilize amylolytic, cellulolytic and xylanolytic enzymes on yeast cell surface for the production of fuel ethanol from biomass. This review gives an insight in to the recent technological developments in the production of bioenergy, i.e, bioethanol using surface engineered yeast.

Role of Immunostimulatory Molecules in Poultry Vaccines by Sachin Kumar, Monika Koul, Anant Rai (235-241).
Immunization by vaccination is the most suitable and safest method for preventing infectious diseases in the poultry worldwide. Vaccines alone cannot effectively protect birds from variety of pathogens under field conditions. The combined use of potent immunostimulants in vaccines is an alternative to increase the efficacy of vaccines that can be achieved by the development of better adjuvant. One such adjuvant is cytokine; cytokines have been used extensively as adjuvant in vaccines and are responsible for the type and extent of an immune response following vaccination. Although the innate immune system in birds is not fully characterized but their immune system is very much similar to that of mammals, and moreover with the recent discovery of a number of avian cytokine genes it is now possible to study their effectiveness in enhancing the immune response during vaccination. This review focuses on the recent studies and developments involving the role of immunomodulating agents especially cytokines of avian origin in poultry vaccines.

Green Biofactories: Recombinant Protein Production in Plants by Adil Ahmad, Eridan O. Pereira, Andrew J. Conley, Alex S. Richman, Rima Menassa (242-259).
Until recently, low accumulation levels have been the major bottleneck for plant-made recombinant protein production. However, several breakthroughs have been described in the past few years allowing for very high accumulation levels, mainly through chloroplast transformation and transient expression, coupled with subcellular targeting and protein fusions. Another important factor influencing our ability to use plants for the production of recombinant proteins is the availability of quick and simple purification strategies. Recent developments using oleosin, zein, ELP and hydrophobin fusion tags have shown promise as efficient and cost-effective methods for nonchromatographic separation. Furthermore, plant glycosylation is a major barrier to the parenteral administration of plantmade biopharmaceuticals because of potential immunogenicity concerns. A major effort has been invested in humanizing plant glycosylation, and several groups have been able to reduce or eliminate immunogenic glycans while introducing mammalian-specific glycans. Finally, biosafety issues and public perception are essential for the acceptance of plants as bioreactors for the production of proteins. Over recent years, it has become clear that food and feed plants carry an inherent risk of contaminating our food supply, and thus much effort has focused on the use of non-food plants. Presently, Nicotiana benthamiana has emerged as the preferred host for transient expression, while tobacco is most frequently used for chloroplast transformation. In this review, we focus on the main issues hindering the economical production of recombinant proteins in plants, describing the current efforts for addressing these limitations, and we include an extensive list of recent patents generated with the intention of solving these limitations.