scholarly journals Molecular farming on rescue of pharma industry for next generations

2018 ◽  
Author(s):  
Khaled Moustafa ◽  
Jocelyne Trémouillaux-Guiller
Keyword(s):  
Recombinant Proteins ◽  
Protein Extraction ◽  
Molecular Farming ◽  
Proper Function ◽  
Protein Accumulation ◽  
Post Translational Modifications ◽  
Extraction And Purification ◽  
Animal Pathogens ◽  
Transformation Methods ◽  
Traditional Approaches

Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.

Visualising intrinsic disorder and conformational variation in protein ensembles

2014 ◽  
Vol 169 ◽  
pp. 179-193 ◽  
Author(s):  
Julian Heinrich ◽  
Michael Krone ◽  
Seán I. O'Donoghue ◽  
Daniel Weiskopf
Keyword(s):  
Intrinsic Disorder ◽  
Molecular Graphics ◽  
Post Translational Modifications ◽  
3D Structures ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Wide Range ◽  
Conformational Variations ◽  
Protein Ensembles ◽  
Traditional Approaches

Intrinsically disordered regions (IDRs) in proteins are still not well understood, but are increasingly recognised as important in key biological functions, as well as in diseases. IDRs often confound experimental structure determination—however, they are present in many of the available 3D structures, where they exhibit a wide range of conformations, from ill-defined and highly flexible to well-defined upon binding to partner molecules, or upon post-translational modifications. Analysing such large conformational variations across ensembles of 3D structures can be complex and difficult; our goal in this paper is to improve this situation by augmenting traditional approaches (molecular graphics and principal components) with methods from human–computer interaction and information visualisation, especially parallel coordinates. We present a new tool integrating these approaches, and demonstrate how it can dissect ensembles to reveal functional insights into conformational variation and intrinsic disorder.

scholarly journals Designing a new generation of expression toolkits for engineering of green microalgae; robust production of human interleukin-2

Bioimpacts ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 259-268
Author(s):  
Jaber Dehghani ◽  
Khosro Adibkia ◽  
Ali Movafeghi ◽  
Mohammad M. Pourseif ◽  
Yadollah Omidi
Keyword(s):  
Recombinant Proteins ◽  
Interleukin 2 ◽  
Cost Effective ◽  
Total Soluble Protein ◽  
Post Translational Modifications ◽  
Unstable Gene ◽  
Protein Synthesis Machinery ◽  
Wide Range ◽  
High Transformation ◽  
New Generation

Introduction: Attributable to some critical features especially the similarity of the protein synthesis machinery between humans and microalgae, these microorganisms can be utilized for the expression of many recombinant proteins. However, low and unstable gene expression levels prevent the further development of microalgae biotechnology towards protein production. Methods: Here, we designed a novel "Gained Agrobacterium-2A plasmid for microalgae expression" (named GAME plasmid) for the production of the human interleukin-2 using three model microalgae, including Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina. The GAME plasmid harbors a native chimeric hsp70/Int-1/rbcS2 promoter, the microalgae specific Kozak sequence, a novel hybrid 2A peptide, and Int-1 and Int-3 of the rbcS2 gene in its expression cassette. Results: The obtained data confirmed that the GAME plasmid can transform the microalgae with high transformation frequency. Molecular and proteomic analyses revealed the stable and robust production of the hIL-2 by the GAME plasmid in the microalgae. According to the densimetric analysis, the microalgae can accumulate the produced protein about 0.94% of the total soluble protein content. The ELISA data confirmed that the produced hIL-2 possesses the same conformation pattern with the acceptable biological activity found naturally in humans. Conclusion: Most therapeutic proteins need post-translational modifications for their correct conformation, biological function, and half-life. Accordingly, microalgae could be considered as a cost-effective and more powerful platform for the production of a wide range of recombinant proteins such as antibodies, enzymes, hormones, and vaccines.

scholarly journals How to approach heterogeneous protein expression for biotechnological use: An overview

2017 ◽  
Vol 16 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Daniela Jamrichová ◽  
Lenka Tišáková ◽  
Veronika Jarábková ◽  
Andrej Godány
Keyword(s):  
Protein Purification ◽  
Recombinant Proteins ◽  
Post Translational Modifications ◽  
Test Protein ◽  
Eukaryotic Gene ◽  
Common Problems ◽  
Escherichia Coli Expression ◽  
The Individual ◽  
Selection Of

AbstractProduction of recombinant proteins in Escherichia coli expression systems has shown many advantages, as well as disadvantages, especially for biotechnological and other down-stream applications. The choice of an appropriate vector depends on the gene, to be cloned for purification procedures and other analyses. Selection of a suitable production strain plays an important role in the preparation of recombinant proteins. The main criteria for the selection of the host organism are the properties of the recombinant produced protein, its subsequent use and the total amount desired. The most common problems in eukaryotic gene expression and recombinant proteins purification are, for instance, post-translational modifications, formation of disulphide bonds, or inclusion bodies. Obtaining a purified protein is a key step enabling further characterization of its role in the biological system. Moreover, methods of protein purification have been developed in parallel with the discovery of proteins and the need for their studies and applications. After protein purification, and also between the individual purification steps, it is necessary to test protein stability under different conditions over time. Shortly, all the essential points have been briefly discussed, which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source and expressed heterogeneously in prokaryotic production system.

SCREENING OF CHLOROPLAST PROMOTERS FOR HEVEA BRASILIENSIS CHLOROPLAST TRANSFORMATION

2015 ◽  
Vol 77 (24) ◽  
Author(s):  
Anas Akmal Ag. Ismail ◽  
Zaima Azira Zainal Abidin ◽  
Zarina Zainuddin
Keyword(s):  
Recombinant Protein ◽  
Plant Species ◽  
Recombinant Proteins ◽  
Hevea Brasiliensis ◽  
Agronomic Traits ◽  
Chloroplast Transformation ◽  
Foreign Protein ◽  
Protein Accumulation ◽  
Nuclear Transformation ◽  
Single Chain

In recent years, the growth in the use of recombinant proteins has grown tremendously. With the aid of the advances in DNA recombinant biotechnology, molecular farming in plants has been applied to meet this increasing demand where plants have emerged as one of the most promising general production platforms for recombinant proteins. Hevea brasiliensis is one of the main commodities in Malaysia and widely cultivated species for commercial production of latex. This important plant has been used to express recombinant proteins such as a single-chain variable fragment (scFv) antibody against the coat protein of Streptococcus gordonii (an oral dental bacterium), human serum albumin and human atrial natriuretic. The genes that encodes for the recombinant proteins were targeted into the nucleus genome of Hevea but the proteins were expressed in low concentration. Generating transgenic plant using chloroplast transformation offers many advantages in comparison to nuclear transformation and many researches have been made to apply this strategy to enhance agronomic traits or produce recombinant protein in several plant species. Since chloroplast is highly polyploidy, it allows high-level foreign protein expression. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts, the aim of this study is to screen a number of chosen endogenous Hevea chloroplast promoters to drive the expression of the reporter gene, uidA for Hevea specific chloroplast transformation vector. Three promoters were chosen for this experiment which are; rbcL, psbA and rrn16 promoters. The putative regions of these promoters were derived from the chloroplast genome sequence of Hevea. Analyses of the three putative promoter regions using multiple sequence alignment with comparable regions from other plant species show significant sequence homology. Further analyses of the putative regions using in-vitro transcription are planned for future study. It is hoped that with the development of an optimized expression vector will allow high expression of valuable recombinant protein in the chloroplast of Hevea.

Rapid production of therapeutic proteins using plant system

2017 ◽  
Vol 2 (2) ◽  
pp. 95 ◽  
Author(s):  
Sathish Kumar Ramalingam ◽  
G. Iyappan ◽  
S. Hari Priya ◽  
K. Kadirvelu ◽  
J. Kingston ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Farming ◽  
Transient Gene Expression ◽  
Functional Protein ◽  
Plant Host ◽  
Post Translational Modifications ◽  
Over Expression ◽  
Plant Molecular Farming ◽  
Rapid Production ◽  
Pharmaceutical Industries

Plant molecular farming is simply defined as the production of proteins therapeutics (PT) in plants, which involves transient gene expression in plants and purification of expressed protein to a great scale for diagnosis, treatment and other applications.  This is therapid,economical, safe and reproducible approach for the production of PTas compared to bacterial and mammalian systems. Protein yield and post-translational modifications are the major roadblocks that can be overcome byhigh expression strategies includes over expression constructs, suitable plant host systems and glycoengineering of proteins. The inherent ability of ideally producing safe, functional protein is the most striking phenomenon recognized by the pharmaceutical industries and developed many therapeutic products within few weeks to meet escalating demands during pandemic/epidemic outbreaks recently

scholarly journals Top-Down Proteomics of Medicinal Cannabis

Proteomes ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 33 ◽  
Author(s):  
Vincent ◽  
Binos ◽  
Rochfort ◽  
Spangenberg
Keyword(s):  
Electron Transfer Dissociation ◽  
Protein Extraction ◽  
Sequence Coverage ◽  
Top Down ◽  
Post Translational Modifications ◽  
Atp Production ◽  
Denatured Protein ◽  
Apical Buds ◽  
Medicinal Cannabis

The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase.

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