Concepts and recent advances in microchip electrophoresis coupled to mass spectrometry: Technologies and applications.

The online coupling of microchip electrophoresis (ME) as a fast, highly efficient, and low-cost miniaturized separation technique to mass spectrometry (MS) as an information-rich and sensitive characterization technique results in ME-MS an attractive tool for various applications. In this paper, we review the basic concepts and latest advances in technology for ME coupled to MS during the period of 2016-2021, covering microchip materials, structures, fabrication techniques, and interfacing to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization-MS. Two critical issues in coupling ME and ESI-MS include the electrical connection used to define the electrophoretic field strength along the separation channel and the generation of the electrospray for MS detection, as well as, a miniaturized ESI-tip. The recent commercialization of ME-MS in zone electrophoresis and isoelectric focusing modes has led to the widespread application of these techniques in academia and industry. Here we summarize recent applications of ME-MS for the separation and detection of antibodies, proteins, peptides, carbohydrates, metabolites, and so on. Throughout the paper these applications are discussed in the context of benefits and limitations of ME-MS in comparison to alternative techniques.

Glyco-Engineering of Plant-Based Expression Systems.

Most secreted proteins in eukaryotes are glycosylated, and after a number of common biosynthesis steps the glycan structures mature in a species-dependent manner. Therefore, human therapeutic proteins produced in plants often carry plant-like rather than human-like glycans, which can affect protein stability, biological function, and immunogenicity. The glyco-engineering of plant-based expression systems began as a strategy to eliminate plant-like glycans and produce human proteins with authentic or at least compatible glycan structures. The precise replication of human glycans is challenging, owing to the absence of a pathway in plants for the synthesis of sialylated proteins and the necessary precursors, but this can now be achieved by the coordinated expression of multiple human enzymes. Although the research community has focused on the removal of plant glycans and their replacement with human counterparts, the presence of plant glycans on proteins can also provide benefits, such as boosting the immunogenicity of some vaccines, facilitating the interaction between therapeutic proteins and their receptors, and increasing the efficacy of antibody effector functions. Graphical Abstract Typical structures of native mammalian and plant glycans with symbols indicating sugar residues identified by their short form and single-letter codes. Both glycans contain fucose, albeit with different linkages.

Overexpression of the recombinant human interferon-beta (rhIFN-ß) gene in tobacco chloroplasts.

Chloroplast genetic engineering is a convenient method for the production of recombinant proteins by increasing the expression level of transgenes. Interferon-beta (IFN-ß) is a member of type I interferons that possess some pharmaceutical properties. The present study aimed to investigate the overexpression and production of the recombinant human IFN-ß gene (rhIFN-ß) in the tobacco chloroplast genome. For this purpose, a codon-optimized rhIFN-ß was transferred to the pVSR326 plastid vector containing the aadA gene as a selectable marker. The rhIFN-ß gene was then successfully introduced into the tobacco chloroplast genome by using a gene gun. The integration of the rhIFN-ß gene into the chloroplast genome and the homoplasmy of the T1 progeny were confirmed by PCR and Southern blot analysis, respectively. RT-PCR and western blot analyses confirmed the transcription and translation of the rhIFN-ß gene, respectively. An enzyme-linked immunosorbent assay (ELISA) showed that the rhIFN-ß protein in transplastomic plants comprised approximately 2.4% of total soluble protein (TSPs). The bioassay confirmed that the rhIFN-ß protein expressed in the tobacco chloroplast had a relatively high biological activity (2.9 × 104 IU/ml) and protected human amnionic cells against the vesicular stomatitis virus (VSV). On the basis of these findings, it can be concluded that plastid transformation can serve as an operative method for the production of pharmaceutical recombinant proteins.

Identification, repair and characterization of a benzyl alcohol-inducible promoter for recombinant proteins overexpression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial organism for the production of a variety of biological commodities. We discovered a promoter encoded by the gene NCgl2319 in C. glutamicum, which could be induced by benzyl alcohol, could be used as an efficient tunable expression system. In initial attempts, this promoter failed to function in a recombinant expression system. This was remedied by extending the original genetic context of the promoter, generating a new version Pcat-B. The Pcat-B transcription initiation site, its critical active regions, and its effect of inducers were fully characterized resulting in tunable expression. This approach proved to be very efficient in producing a pharmaceutical protein, N-terminal pro-brain natriuretic peptide (NT-proBNP). Production of approximately 440.43 mg/L NT-proBNP was achieved with the Pcat-B expression system demonstrating its application for controllable pharmaceutical protein production in C. glutamicum.

A New Plant Expression System for Producing Pharmaceutical Proteins.

In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.

[Cell-free synthetic biology: an emerging strategy torevolutionize the biomedical industry].

Cell-free synthetic biology system can perform biological transcription and translation process in vitro. Because of its advanced features, such as flexible openness, easy control, short expression time and high tolerance to cytotoxicity, this systemhas been successfully used to synthesize proteins that are difficult to express in cells. With the continuous development of cell-free biosensing technology and the lyophilization technology, its applications have widely expanded into many biomedical fields. This review discusses the current research progress of cell-free synthetic biology system in on-demand biopharmaceutical synthesis, portable diagnostics, and others. Further development of the system can lead to even more complicated synthesis of therapeutic proteins with post-translational modifications and evolution of different cell-free biosensors with high sensitivity. Cell-free synthetic biology as an emerging engineering strategy can be a better means applied to high-throughput screening of pharmaceutical proteins, detection of new pathogens, and other important health-care fields in the future.

Effects of air temperature before harvest on the concentration of human adiponectin in transgenic strawberry fruits.

Transgenic ever-bearing strawberry (Fragaria×ananassa Duch. 'HS 138') was cultivated in a closed plant production system to produce functional proteins that can enhance human immune functions. We investigated the effects of air temperature before harvest on fruit growth and the concentration of human adiponectin (hAdi) at harvest in transgenic strawberry. During the different stages of maturity (mature white and immature green stages), hAdi-expressing plants were exposed to four air temperature treatments (15, 20, 25, and 30°C) under 24-h illumination provided by fluorescent lamps. Fruits were harvested at the mature red stage. The number of days to the mature red stage decreased with increasing air temperature, being the least at 30°C. Fruit total soluble protein (TSP) concentration increased with decreasing air temperature, particularly at 15°C, whereas fruit hAdi concentration tended to be higher under the 30°C treatment than under any other of the temperature treatments. There was no significant relationship between fruit fresh weight at harvest time and hAdi concentration within treatments, nor between the number of days to harvest and hAdi or TSP concentration. Although there were no significant differences in fruit hAdi content among treatments, hAdi production rate was the highest at 30°C because of the shortest duration to harvest. These results indicate that a higher air temperature promoted fruit maturation and accelerated the production of functional hAdi proteins in the fruit. For hAdi-expressing strawberry plants, exposure to 30°C may reduce energy consumption (lighting and air conditioning) for functional protein production under controlled environments.

Cell-free synthetic biology: an emerging strategy torevolutionize the biomedical industry / 生物工程学报

Cell-free synthetic biology system can perform biological transcription and translation process in vitro. Because of its advanced features, such as flexible openness, easy control, short expression time and high tolerance to cytotoxicity, this systemhas been successfully used to synthesize proteins that are difficult to express in cells. With the continuous development of cell-free biosensing technology and the lyophilization technology, its applications have widely expanded into many biomedical fields. This review discusses the current research progress of cell-free synthetic biology system in on-demand biopharmaceutical synthesis, portable diagnostics, and others. Further development of the system can lead to even more complicated synthesis of therapeutic proteins with post-translational modifications and evolution of different cell-free biosensors with high sensitivity. Cell-free synthetic biology as an emerging engineering strategy can be a better means applied to high-throughput screening of pharmaceutical proteins, detection of new pathogens, and other important health-care fields in the future.

Production of pharmaceutical proteins by transgenic animals.

Proteins are involved in a majority of the biochemical events that take place in all living organisms. Protein synthesis is directed by genes. All genes contain two major DNA regions. The region containing the genetic message proper (the 'coding region') is preceded by a regulatory region ('the promoter'), which determines when and in which organs a given gene must produce the corresponding protein. The techniques of genetic engineering allow the association of the coding region from one gene with the regulatory region from another gene. The expression of these recombinant genes may be achieved in cultured cells, in transgenic animals or in plants. This leads to the production of the corresponding proteins, including pharmaceutical proteins. Milk from transgenic animals is one potential source of pharmaceutical proteins. To achieve this, the promoters from milk protein genes are bound to DNA fragments containing the coding region of the genes of interest. The desired proteins are then taken from the milk and purified. Two human pharmaceutical proteins are on the market and about 20 projects are in development. One of the proteins produced in milk, antithrombin III, is an anticoagulant and the other, human C1-esterase inhibitor, is an anti-inflammatory. Several human proteins have been produced in the egg white of transgenic chickens and one has been approved by the United States Federal Drug Administration. This process has also been used to modify antibodies in cows. The genes that code for antibodies in the cow were deleted and replaced by human antibody genes. These cows, immunised by various antigens, then secreted purely human antibodies in their blood. Antibodies from such cows were able to attenuate the effects of Ebola virus in human patients.

Les protéines sont des acteurs moléculaires majeurs des organismes vivants. Leur synthèse résulte du décodage des gènes. Essentiellement, tous les gènes contiennent deux régions d'ADN majeures. La région contenant le message génétique proprement dit (région codante) est précédée d'une région de régulation (le promoteur) qui détermine quand et dans quels organes un gène donné doit produire la protéine correspondante. Les techniques de génie génétique permettent l'association de la région codante d'un gène donné à une région régulatrice d'un autre gène. L'expression des gènes recombinants peut être réalisée dans des cellules cultivées, dans des animaux ou des plantes transgéniques. Ceci conduit à la production des protéines correspondantes comprenant des protéines pharmaceutiques. Le lait d'animaux transgéniques a été retenu comme l'une des sources potentielles de protéines pharmaceutiques. Pour atteindre cet objectif, les promoteurs des gènes des protéines du lait sont liés à des fragments d'ADN contenant les gènes d'intérêt. Les protéines sont ainsi retrouvées dans le lait et purifiées. Deux protéines pharmaceutiques humaines sont sur le marché et une vingtaine de projets sont en cours de développement. L'une des protéines produites dans le lait, l'antithrombine III, est un anticoagulant, tandis que l'autre, l'inhibiteur de la C1-estérase humaine, est un anti-inflammatoire. Plusieurs protéines humaines ont été produites dans du blanc d'oeuf de poulet transgénique et l'une d'entre elles a été approuvée par l'Agence américaine des produits alimentaires et médicamenteux. Cette méthode a également été utilisée pour modifier l'expression génétique des anticorps chez des vaches. Des vaches dont les gènes codant les anticorps ont été inactivés et remplacés par les gènes d'anticorps humains ont été obtenues. Ces vaches immunisées par divers antigènes sécrètent dans leur sang des anticorps purement humains. Certains anticorps de telles vaches ont été capables d'atténuer les effets du virus Ebola chez les patients.

Las proteínas participan en la mayoría de las reacciones bioquímicas que tienen lugar en todos los organismos vivos. La síntesis de proteínas está regida por los genes. Todos los genes contienen dos regiones principales de ADN. La región que contiene el mensaje genético propiamente dicho («región codificante¼) viene precedida de una región reguladora («promotora¼), que determina cuándo y en qué órganos un determinado gen debe expresar la correspondiente proteína. Las técnicas de ingeniería genética permiten asociar entre sí la región codificante de un gen y la región reguladora de otro gen. Estos genes recombinantes pueden ser llevados a expresarse en cultivos celulares, animales transgénicos o plantas, lo que resulta en la producción de las correspondientes proteínas, entre ellas proteínas farmacéuticas. La leche de animales transgénicos es una posible fuente de proteínas farmacéuticas. Para obtenerlas se unen las secuencias promotoras de los genes de proteínas lácteas con fragmentos de ADN que contienen la región codificante de los genes que interesan. Después se extraen de la leche y se purifican las proteínas deseadas. Ahora mismo hay dos proteínas farmacéuticas humanas que ya están comercializadas, además de unos 20 proyectos en curso. Una de las proteínas fabricadas en leche, la antitrombina III, es un anticoagulante, y la otra, inhibidora de la C1 esterasa humana, un antiinflamatorio. Varias proteínas humanas, una de ellas ya aprobada por la Administración de Alimentos y Medicamentos de los Estados Unidos, han sido producidas en la clara de huevo de gallinas transgénicas. Este proceso también ha sido utilizado para modificar anticuerpos en vacas. Los genes que codifican los anticuerpos de la vaca son suprimidos y sustituidos por genes de anticuerpos humanos. Estas vacas, expuestas a varios antígenos, secretan y envían al torrente sanguíneo anticuerpos humanos puros. Con los anticuerpos obtenidos a partir de tales vacas se pudieron atenuar los efectos del virus Ebola en pacientes humanos.

In Silico Study to Develop a Lectin-Like Protein from Mushroom Agaricus bisporus for Pharmaceutical Application.

A lectin-like protein of unknown function designated as LSMT was recently discovered in the edible mushroom Agaricus bisporus. The protein shares high structural similarity to HA-33 from Clostridium botulinum (HA33) and Ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which have been developed as drug carrier and anti-cancer, respectively. These homologous proteins display the ability to penetrate the intestinal epithelial cell monolayer, and are beneficial for oral administration. As the characteristics of LSMT are unknown, a structural study in silico was performed to assess its potential pharmaceutical application. The study suggested potential binding to target ligands such as HA-33 and CNL although the nature, specificity, capacity, mode, and strength may differ. Further molecular docking experiments suggest that interactions between the LSMT and tested ligands may take place. This finding indicates the possible use of the LSMT protein, initiating new research on its use for pharmaceutical purposes.

Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin).

Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90 days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis.

Progress in medicinal research and applications of oral recombinant lactic acid bacteria / 中国药学杂志

OBJECTIVE: To elaborate the advantages, immunological mechanisms of medicinal effects, influencing factors as well as applications of recombinant lactic acid bacteria (rLAB). In addition, the issues of their development are also discussed.

The use of rice seeds to produce human pharmaceuticals for oral therapy.

Rice (Oryza sativa L.) is the major staple food consumed by half of the world's population. Rice seeds have gained recent attention as bioreactors for the production of human pharmaceuticals such as therapeutic proteins or peptides. Rice seed production platforms have many advantages over animal cell or microbe systems in terms of cost-effectiveness, scalability, safety, product stability and productivity. Rice seed-based human pharmaceuticals are expected to become innovative therapies as edible drugs. Therapeutic proteins can be sequestered within natural cellular compartments in rice seeds and protected from harsh gastrointestinal environments. This review presents the state-of-the-art on the construction of gene cassettes for accumulation of pharmaceutical proteins or peptides in rice seeds, the generation of transgenic rice plants, and challenges involved in the use of rice seeds to produce human pharmaceuticals.