Root-secreted bitter triterpene modulates the rhizosphere microbiota to improve plant fitness

Production of foreign proteins using plastid transformation.

Myocardial infarction and ischemic stroke are the most frequent causes of death or disability worldwide. Due to their ability to dissolve blood clots, the thrombolytics are frequently used for their treatment. Improving the effectiveness of thrombolytics for clinical uses is of great interest. The knowledge of the multiple roles of the endogenous thrombolytics and the fibrinolytic system grows continuously. The effects of thrombolytics on the alteration of the nervous system and the regulation of the cell migration offer promising novel uses for treating neurodegenerative disorders or targeting cancer metastasis. However, secondary activities of thrombolytics may lead to life-threatening side-effects such as intracranial bleeding and neurotoxicity. Here we provide a structural biology perspective on various thrombolytic enzymes and their key properties: (i) effectiveness of clot lysis, (ii) affinity and specificity towards fibrin, (iii) biological half-life, (iv) mechanisms of activation/inhibition, and (v) risks of side effects. This information needs to be carefully considered while establishing protein engineering strategies aiming at the development of novel thrombolytics. Current trends and perspectives are discussed, including the screening for novel enzymes and small molecules, the enhancement of fibrin specificity by protein engineering, the suppression of interactions with native receptors, liposomal encapsulation and targeted release, the application of adjuvants, and the development of improved production systems.

Structural Biology and Protein Engineering of Thrombolytics.

Transgenic plant-derived vaccines comprise a new type of bioreactor that combines plant genetic engineering technology with an organism's immunological response. This combination can be considered as a bioreactor that is produced by introducing foreign genes into plants that elicit special immunogenicity when introduced into animals or human beings. In comparison with traditional vaccines, plant vaccines have some significant advantages, such as low cost, greater safety, and greater effectiveness. In a number of recent studies, antigen-specific proteins have been successfully expressed in various plant tissues and have even been tested in animals and human beings. Therefore, edible vaccines of transgenic plants have a bright future. This review begins with a discussion of the immune mechanism and expression systems for transgenic plant vaccines. Then, current advances in different transgenic plant vaccines will be analyzed, including vaccines against pathogenic viruses, bacteria, and eukaryotic parasites. In view of the low expression levels for antigens in plants, high-level expression strategies of foreign protein in transgenic plants are recommended. Finally, the existing safety problems in transgenic plant vaccines were put forward will be discussed along with a number of appropriate solutions that will hopefully lead to future clinical application of edible plant vaccines.

/pdf/recent-advances-and-safety-issues-of-transgenic-plant-31ubvo48rx.pdf

Recent advances and safety issues of transgenic plant-derived vaccines

Summary
Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2011.00612.x

Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes.

Synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit–HN-neutralizing epitope fusion protein was expressed in E. coli and tobacco chloroplasts. Bacterial and chloroplastic recombinant LTB–HN-neutralizing epitope (LTB-HNE) fusion proteins showed the pentameric structures by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a strong affinity for GM1-ganglioside. Bacterial and chloroplastic recombinant LTB-HNE was detected by Western blot analysis using polyclonal antibodies to HN-neutralizing epitope. Insertion of the gene encoding LTB-HNE protein into the chloroplast genomic DNA of spectinomycin-resistant plants was confirmed by polymerase chain reaction. The presence of the LTB-HNE specific transcript in the total RNA of transgenic plant leaves was verified by reverse transcriptase polymerase chain reaction. The highest level of expression of recombinant LTB-HNE fusion proteins in the leaves of transplastomic plants was about 0.5% of the total soluble protein. Growth rates, flowering, and seed setting were not affected, and the cassette for homologous integration and gene expression in the transplastomic T1 plant was subsequently inherited.

Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin–Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating the transition from plasminogen to plasmin in blood vessels. The recombinant tissue-plasminogen activator (rTPA) produced in plants is a soluble protein derived from human t-PA. In order to produce rTPA protein in plants, a DNA fragment encoding the t-PA protein was cloned into a plant binary plasmid that included the cauliflower mosaic virus 35S promoter, a tobacco etch virus (TEV) leader sequence, an N-terminal signal peptide of the alfala glucose-regulated endoplasmic reticular protein, and an 35S terminator. The insertion of the rTPA gene in genomic DNA of phospinothricin-resistant tobacco plants was confirmed by polymerase chain reaction (PCR). The presence of the rTPA-specific transcript in the total RNAs of transgenic plants leaves was verified by reverse transcription (RT)-PCR. Immunoblot analysis of the transformed tobacco leaves revealed single bands of 68-kDa rTPAs. Enzyme-linked immunosorbent assay experiments demonstrated that the highest level of rTPA expression was 0.0014% of the total soluble protein in tobacco plants. Fibrinolysis of rTPA was confirmed by fibrin plate assay and zymography. These studies demonstrate that plants can be employed as bioreactor systems for the production of an enzymatically active t-PA with properties similar to the recombinant t-PA produced by mammalian cells.

Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

Cotyledon explants of two Oriental melons produced hairy roots when cultured on Murashige and Skoog basal medium after infection by the Agrobacterium
 rhizogenes. Hairy roots were produced from the wounded surface of the cotyledon explants of Cucumis melo L. cv. Geumssaragi-euncheon on Murashige and Skoog selective medium and 86% of the GUS stained hairy roots were positive for the expression of beta-glucuronidase. The insertion of the gfp-gus fusion gene in the genomic DNA and the presence of the gfp-gus-specific transcript in the total RNAs of transgenic hairy roots were confirmed by PCR and RT-PCR, respectively. An immunoblot analysis of the transgenic hairy root extract revealed 97 kDa single bands coincident with the molecular weight of the GFP-GUS fusion proteins. ELISA demonstrated that the highest level of GFP-GUS fusion protein expression was 0.47% of the total soluble protein in a transgenic hairy root. The MS medium showed the fastest growth among three media types tested. Infection of the hairy roots with a root-knot nematode resulted in the development of a mature egg mass about 4–5 weeks after inoculation. The highest number of egg mass was obtained on the hairy roots cultured in SH medium containing 0.3% agar.

Hairy root induction in Oriental melon (Cucumis melo) by Agrobacterium rhizogenes and reproduction of the root-knot nematode (Meloidogyne incognita)

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent important in fibirn clot lysis. T-PA causes fibirn-specific plasminogen activation. Six binary vectors harboring t-PA and its derivative genes were cloned and expressed in transgenic alfalfa plants. The insertion of the t-PA and its derivative genes in genomic DNA of alfalfa plants was confirmed by PCR. The presence of the t-PA and its derivative transcripts in total RNAs of the transgenic alfalfa leaves was verified by RT-PCR. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was / total soluble protein (mg) in alfalfa plants. The amount of recombinant t-PA and its derivative proteins in transgenic plants was estimated to range from 9.7 to / total soluble proteins (mg). Western blot analysis of the transformed alfalfa leaves revealed bands of approximately 68-kDa recombinant t-PA and its derivative proteins. The fibrinolysis of recombinant t-PA and its derivative proteins was confirmed by a fibrin plate assay (range from 3.2 to 8.1 cm). The results presented provide information for the development of an additional production of recombinant human proteins having pharmaceutical applications using transgenic plants.

http://www.ndsl.kr/soc_img/society/kspbt/SMSMCM/2009/v36n1/SMSMCM_2009_v36n1_30.pdf

Expression of tissue-type plasminogen activator and its derivative proteins in transgenic alfalfa plants

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Hyo-Kyung Pak

Author Tools

Chat about Author

Papers

Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin–Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

Hairy root induction in Oriental melon (Cucumis melo) by Agrobacterium rhizogenes and reproduction of the root-knot nematode (Meloidogyne incognita)

Expression of tissue-type plasminogen activator and its derivative proteins in transgenic alfalfa plants