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Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii
Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news

Transient Protein Expression by Agroinfiltration in Lettuce
Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed “deconstructed” viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news

Virus-Derived Vectors for the Expression of Multiple Proteins in Plants
This chapter constitutes a practical guide to using the “pEAQ” vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV-HT, is used. The expression vector pEAQ-HT combines the user-friendly pEAQ plasmid with CPMV-HT to provide a system for high-level expression of protei...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news

Multigene Engineering in Rice Using High-Capacity Agrobacterium tumefaciens BIBAC Vectors
The high-capacity binary bacterial artificial chromosome (BIBAC) vector system permits the insertion of large fragments of DNA, up to 150 kb, into plants via Agrobacterium-mediated transformation. Here, we describe an optimized protocol for transformation of japonica rice (Oryza sativa L.) using this system. Calli derived from mature embryos are transformed using Agrobacterium strain LBA4404 that carries the BIBAC vector and the super-virulent helper plasmid pCH32. Transformed calli are then regenerated using optimized media and tested for transgene integration by PCR, GUS assay, and Southern blot analyses. (Source: Spring...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news

Chloroplast-Based Expression of Recombinant Proteins by Gateway® Cloning Technology
We describe the steps required for cloning, biolistic transformation, identification, and regeneration of transplastomic plant lines and Western blot analysis. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news

Extraction and Purification of Phlorotannins from Brown Algae
We describe here a simple procedure based on the use of water-organic solvent mixtures for the extraction of phlorotannins. Crude extracts are semi-purified and fractionated by separating methods based on both the polarity and the molecular size of compounds. Phenols are then quantified by the Folin-Ciocalteu method and their radical-scavenging activity is characterized using the DPPH test. All along the purification process of phenolic compounds, the efficiency of separation is assessed by 1H-NMR. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Extraction and Analysis of Mycosporine-Like Amino Acids in Marine Algae
Marine organisms use mycosporine-like amino acids (MAAs) as biological sunscreens for the protection from damaging ultraviolet (UV) radiation and the prevention of oxidative stress. MAAs have been discovered in many different marine and freshwater species including cyanobacteria, fungi, and algae, but also in animals like cnidarian and fishes. Here, we describe a general method for the isolation and characterization of MAA compounds from red algae and symbiotic dinoflagellates isolated from coral hosts. This method is also suitable for the extraction and analyses of MAAs from a range of other algal and marine biota. (Sourc...
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Extraction and Purification of R-phycoerythrin from Marine Red Algae
This chapter focuses on the recovery of an R-Phycoerythrin (R-PE)-enriched fraction from marine algae. Since R-PE is a proteinaceous pigment, we have developed a simple and rapid two-step method devoted to the extraction and purification of R-PE from marine red algae. Here we describe a phosphate buffer extraction followed by anion exchange chromatography carried on a DEAE Sepharose Fast Flow column. To ensure the quality and quantity of R-PE recovery, we also indicate different methods to monitor each fraction obtained, such as spectrophotometric indicators, gel filtration, and SDS-PAGE analysis. (Source: Springer protoco...
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Extraction and Enrichment of Protein from Red and Green Macroalgae
Macroalgae, in particular red and green species, are gaining interest as protein-rich foods for human consumption and sources of proteinaceous biofunctional peptide ingredients. During protein extraction the starting raw material, the cell disruption method utilized and the reagents employed have a major effect on the yield of protein recovered. A method is described herein for extraction and semi-purification of food-grade aqueous and alkaline soluble proteins from red and green macroalgae. Dried milled macroalgae are disrupted by osmotic shock with subsequent removal of aqueous soluble proteins by centrifugation. Alkalin...
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Spectrophotometric Assays of Major Compounds Extracted from Algae
This chapter describes spectrophotometric assays of major compounds extracted from microalgae and macroalgae, i.e., proteins, carbohydrates, pigments (chlorophylls, carotenoids, and phycobiliproteins) and phenolic compounds. In contrast to other specific analytical techniques, such as high pressure liquid chromatography (HPLC) or mass spectrometry (MS), commonly applied to purified extracts to reveal more detailed composition and structure of algal compound families, these assays serve as a first assessment of the global contents of extracts. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Structure and Function of Macroalgal Natural Products
Since the initial discovery of marine phyco-derived secondary metabolites in the 1950s there has been a rapid increase in the description of new algal natural products. These metabolites have multiple ecological roles as well as commercial value as potential drugs or lead compounds. With the emergence of resistance to our current arsenal of drugs as well as the development of new chemotherapies for currently untreatable diseases, new compounds must be sourced. As outlined in this chapter algae produce a diverse range of chemicals many of which have potential for the treatment of human afflictions. (Source: Springer protoco...
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Protocol for Assessing Antifouling Activities of Macroalgal Extracts
The development of novel environmentally friendly antifouling (AF) solutions is a very active field in fundamental and applied research. An attractive option in producing such material resides in biomimetic studies: living organisms have evolved well-adapted structures and materials over geological times through natural selection. In this chapter, we explain the experimental procedure to be followed for the preparation of macroalgal extracts and to assess their AF efficiency towards key species. All bioassays described here have the advantage of being fast, reliable, and standardized. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Screening of a Marine Algal Extract for Antifungal Activities
Over the past few years algal extracts have become increasingly interesting to the scientific community due to their promising biological properties. Phlorotannin extracts are particularly attractive partly due to their reported antifungal activity against several yeast and dermatophyte strains. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

Disk Diffusion Assay to Assess the Antimicrobial Activity of Marine Algal Extracts
Marine algae are a relatively untapped source of bioactive natural products, including those with antimicrobial activities. The ability to assess the antimicrobial activity of cell extracts derived from algal cultures is vital to identifying species that may produce useful novel antibiotics. One assay that is used widely for this purpose is the disk diffusion assay due to its simplicity, rapidity, and low cost. Moreover, this assay gives output data that are easy to interpret and can be used to screen many samples at once irrespective of the solvent used during preparation. In this chapter, a step-by-step protocol for perf...
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news

In Vitro Protocols for Measuring the Antioxidant Capacity of Algal Extracts
In the last decade a large amount of research has been directed at targeting algal resources for biologically active molecules. High-throughput in vitro antioxidant assays are routinely used to screen for biologically active compounds present in algal extracts when the requirement is to identify samples for progression to more detailed biological scrutiny. Whilst a myriad of antioxidant assays have been developed, this present chapter aims to give step-by-step practical guidance on how to carry out some of the most popular and biologically relevant assays at the bench. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - June 26, 2015 Category: Biology Source Type: news