Understanding Marine Mussel Adhesion

In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.     

Mussel byssus and biomolecular materials

Mussel adhesive proteins are remarkable materials that display an extraordinary capability to adhere to substrates underwater. Recent investigations from groups with quite diverse areas of expertise have made substantial progress in the identification of the genes and proteins that are involved in adhesive formation. These discoveries have led to the development of recombinant proteins and synthetic polypeptides that are able to reproduce the properties of mussel adhesives for applications in medicine and biotechnology.     

Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Development of bioadhesives from marine mussels

Mussel adhesive proteins have received increased attention as potential biomedical and environmentally friendly underwater adhesives thanks to their fascinating properties, including strong and flexible adhesion, adhesion to various material substrates, water displacement, that they are harmless to human body, and controlled biodegradability. Several mussel adhesive proteins have been identified and characterized from mussels, and profound biochemical knowledge for mussel adhesions has been accumulated. In addition, a lot of effort has been put into realizing the promise of these bioadhesive materials from marine mussels. Here, progress in the diverse developmental approaches, with particular emphasis on functional production of mussel adhesive proteins, are reviewed.     

重组贻贝足蛋白的研究进展与应用*

贻贝足蛋白是一类通过贻贝足腺分泌的蛋白质复合物,与基质表面发生反应而产生极强的黏附作用。其在海洋环境中具有强黏附能力、可降解性和优秀的生物相容性等优点,因此常被用做生物医药黏合剂。但提取天然蛋白质受原料来源限制,且工艺烦琐导致价格高昂,阻碍了贻贝足蛋白的进一步应用发展。微生物合成的最新进展为贻贝足蛋白的产出提供了一种新思路,并且具有扩大规模生产的意义。主要综述了贻贝足蛋白的基因工程生产方法,总结了重组蛋白在黏附抗污涂层、组织工程材料等领域的应用现状。同时对其研究方向进行了展望,指出重组贻贝足蛋白的进一步发展的关键技术是解析蛋白质的构效和层级结构,在此基础上提高其异源表达水平,以获得更多生物功效性的衍生产品。     

Expression of functional recombinant mussel adhesive protein type 3A in Escherichia coli

Mussel adhesive proteins, including the 20-plus variants of foot protein type 3 (fp-3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. Here we report the novel production of a recombinant Mytilus galloprovincialis foot protein type 3 variant A (Mgfp-3A) fused with a hexahistidine affinity ligand in Escherichia coli and its approximately 99% purification with affinity chromatography. Recombinant Mgfp-3A showed a superior purification yield and better apparent solubility in 5% acetic acid (prerequisites for large-scale production and practical use) compared to those of the previously reported recombinant M. galloprovincialis foot protein type 5 (Mgfp-5). The adsorption abilities and adhesion forces of purified recombinant Mgfp-3A were compared with those of Cell-Tak (a commercial mussel extract adhesive) and recombinant Mgfp-5 using quartz crystal microbalance analysis and modified atomic force microscopy, respectively. These assays showed that the adhesive ability of recombinant Mgfp-3A was comparable to that of Cell-Tak but lower than that of recombinant Mgfp-5. Collectively, these results indicate that recombinant Mgfp-3A may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments.     

Thematic minireview series on circular proteins

Circular proteins have now been discovered in all kingdoms of life and are characterized by their exceptional stability and the diversity of their biological activities, primarily in the realm of host defense functions. This thematic minireview series provides an overview of the distribution, evolution, activities, and biological synthesis of circular proteins. It also reviews approaches that biological chemists are taking to develop synthetic methods for making circular proteins in the laboratory. These approaches include solid-phase peptide synthesis based on an adaption of native chemical ligation technology and recombinant DNA approaches that are amenable to the in-cell production of cyclic peptide libraries. The thioester-mediated native chemical ligation approach mimics, to some extent, elements of the natural biosynthetic reaction, which, for disulfide-rich cyclic peptides, appears to involve asparaginyl endopeptidase-mediated processing from larger precursor proteins.     

Marine adhesive proteins: natural composite thermosets

Marine environments are severely challenging for the performance and durability of synthetic adhesives. Factors commonly associated with adhesive failure are weak boundary layers (water, oxides), adhesive erosion and swelling. For many permanently attached marine organisms such as barnacles, mussels, oysters, etc., however, underwater adhesion is 'business-as-usual'. Knowledge about the chemistry and bioprocessing of these marine adhesives will provide profound insights for the evolution of a new generation of environmentally safe, water-resistant adhesives. Despite their apparent structural diversity, marine adhesives are essentially analogous to composite thermosets, that is, the adhesive consists of fibre, filler and catalyst molecules that are dispersed in a cross-linked resin rendering it resistant to heat and solvents. The fibres and fillers in these composites are variable. e.g. collagen, fibroin, chitin present as fibres, and sand, shell, air and water present as fillers. The precured resins of seven organisms including members of the Mollusca, Annelida, and Platyhelminthes have now been isolated and partially sequenced. These are proteins with basic isoelectric points, high levels of the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA), and an extended, flexible conformation. The DOPA functional group in particular is thought to play a key role in (a) the chemisorption of these polymers to surface underwater, and (b) covalent cross-linking or setting of the adhesive, the latter reaction catalysed by the enzyme catecholoxidase. Much more needs to be done to explore the details of the adhesive processing and delivery strategies used by these organisms.     

CHO细胞表达系统启动子

中国仓鼠卵巢（Chinese hamsters ovary,CHO）细胞是目前重组蛋白质生产的首选宿主细胞。利用CHO细胞生产重组蛋白质,启动子是启动转基因转录的关键。核心启动子是RNA聚合酶与转录起始复合物集合的部位,分为集中型和分散型两种类型。目前,CHO细胞常用的启动子为病毒启动子、异源启动子、内源性和诱导性启动子等。也可以利用合成生物学及相关的数据库,人工设计合成启动子及鉴定新型启动子。本文综述了CHO细胞常用的启动子以及人工设计的合成启动子在CHO细胞中重组蛋白质表达方面的进展,为哺乳动物细胞选择合适的启动子,保证蛋白质表达量最大化,并确保长时间表达稳定性提供参考。     

CHO细胞表达系统启动子

中国仓鼠卵巢（Chinese hamsters ovary,CHO）细胞是目前重组蛋白质生产的首选宿主细胞。利用CHO细胞生产重组蛋白质,启动子是启动转基因转录的关键。核心启动子是RNA聚合酶与转录起始复合物集合的部位,分为集中型和分散型两种类型。目前,CHO细胞常用的启动子为病毒启动子、异源启动子、内源性和诱导性启动子等。也可以利用合成生物学及相关的数据库,人工设计合成启动子及鉴定新型启动子。本文综述了CHO细胞常用的启动子以及人工设计的合成启动子在CHO细胞中重组蛋白质表达方面的进展,为哺乳动物细胞选择合适的启动子,保证蛋白质表达量最大化,并确保长时间表达稳定性提供参考。     

Synthetic biology advances and applications in the biotechnology industry: a perspective

Synthetic biology is a logical extension of what has been called recombinant DNA (rDNA) technology or genetic engineering since the 1970s. As rDNA technology has been the driver for the development of a thriving biotechnology industry today, starting with the commercialization of biosynthetic human insulin in the early 1980s, synthetic biology has the potential to take the industry to new heights in the coming years. Synthetic biology advances have been driven by dramatic cost reductions in DNA sequencing and DNA synthesis; by the development of sophisticated tools for genome editing, such as CRISPR/Cas9; and by advances in informatics, computational tools, and infrastructure to facilitate and scale analysis and design. Synthetic biology approaches have already been applied to the metabolic engineering of microorganisms for the production of industrially important chemicals and for the engineering of human cells to treat medical disorders. It also shows great promise to accelerate the discovery and development of novel secondary metabolites from microorganisms through traditional, engineered, and combinatorial biosynthesis. We anticipate that synthetic biology will continue to have broadening impacts on the biotechnology industry to address ongoing issues of human health, world food supply, renewable energy, and industrial chemicals and enzymes.     

Brave new proteins: what evolution reveals about protein structure.

Synthetic proteins provide important information about the principles of protein structure. They illuminate the processes of natural protein evaluation, but are not limited by these processes. Here, we review studies of several mutant proteins and discuss the general principles that can be derived from them.     

Synthetic paths towards scalaranes: Assembling the scalaranic skeleton and further transformations

The family of scalaranic sesterterpenoids has been known since the early 70s, but they have attracted the attention of synthetic organic chemists only in the last decade. These marine natural products are considered secondary metabolites of marine organisms and have shown promising biological properties. Synthetic approaches towards scalaranes are discussed in the present review. Scalaranes are quite complex compounds, with many chiral centers and their transformations are strongly directed by steric effects of different functional groups attached to the tetracyclic framework. Therefore, methods of assembling the scalaranic skeleton are examined first, followed by the synthesis of natural products or their analogues on the basis of the available tetracyclic template.Dedicated to our teacher Iurii Revenco on the occasion of his 75th birthday     

Basic science through engineering? Synthetic modeling and the idea of biology-inspired engineering

Synthetic biology is often understood in terms of the pursuit for well-characterized biological parts to create synthetic wholes. Accordingly, it has typically been conceived of as an engineering dominated and application oriented field. We argue that the relationship of synthetic biology to engineering is far more nuanced than that and involves a sophisticated epistemic dimension, as shown by the recent practice of synthetic modeling. Synthetic models are engineered genetic networks that are implanted in a natural cell environment. Their construction is typically combined with experiments on model organisms as well as mathematical modeling and simulation. What is especially interesting about this combinational modeling practice is that, apart from greater integration between these different epistemic activities, it has also led to the questioning of some central assumptions and notions on which synthetic biology is based. As a result synthetic biology is in the process of becoming more “biology inspired.”     

Human cells: new platform for recombinant therapeutic protein production

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production.     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Single honeybee silk protein mimics properties of multi-protein silk

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.     

Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin

Mussel adhesive proteins (MAPs) have been considered as potential underwater and medical bioadhesives. Previously, we reported a functional expression of recombinant MAP hybrid fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus, with practical properties in Escherichia coli. In the present work, we introduced the Vitreoscilla hemoglobin (VHb) co-expression strategy to enhance the production levels of hybrid fp-151 since VHb has been successfully used for efficient oxygen utilization in several expression systems, including E. coli. In both batch-type flask and fed-batch-type bioreactor cultures, we found that co-expression of VHb conferred higher cell growth and hybrid fp-151 production. Its positive effects were significantly increased in high cell density bioreactor cultures as the microaerobic environment was more quickly and severely formed. We obtained a approximately 1.9-fold higher (approximately 1 g/L) production of MAP fp-151 from VHb co-expressing cells in fed-batch bioreactor cultures as compared to that from VHb non-expressing cells. Collectively and regardless of the culture type, VHb co-expression strategy was successful in enhancing the production of recombinant mussel adhesive proteins in the E. coli expression system.     

Optimisation of signal peptide for recombinant protein secretion in bacterial hosts

Escherichia coli—the powerhouse for recombinant protein production—is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.