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Find video protocols related to scientific articles indexed in Pubmed.
Agrobacterium tumefaciens mediated transient expression of plant cell wall-degrading enzymes in detached sunflower leaves.
Biotechnol. Prog.
PUBLISHED: 09-03-2014
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For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S-driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA-Based Overexpression Vector) systems. For 6-day leaf incubations, codon-optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two-phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6-fold. Production also increased up to 4.2-fold when the leaf incubation temperature was elevated above the typical temperature, 20C, to 30C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co-expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall-degrading enzymes in sunflower.
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Production and characterization of Acidothermus cellulolyticus endoglucanase in Pichia pastoris.
Protein Expr. Purif.
PUBLISHED: 01-15-2011
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The endoglucanase (E1) from Acidothermus cellulolyticus has been used extensively in cellulase research. The goal of this work was to produce high levels of this enzyme in a system that facilitates purification. A codon-optimized synthetic gene for A. cellulolyticus E1 with a C-terminal histidine tag was cloned into the genome of Pichia pastoris. Strain KM71H expressed the most enzyme, with a yield of 550mg/L culture supernatant. The temperature optimum (80°C) and pH optimum (5.1) of the purified enzyme agree with previously determined values for the enzyme produced in other systems. Michaelis-Menten kinetic parameters were determined, using a fluorescent substrate (methylumbelliferyl-?-d-cellobioside) at various temperatures. This thermostable enzyme can be used in future cellulosic biofuels-related research.
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Bipartite and tripartite Cucumber mosaic virus-based vectors for producing the Acidothermus cellulolyticus endo-1,4-?-glucanase and other proteins in non-transgenic plants.
BMC Biotechnol.
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Using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. The objective of this work was the development of advanced CMV-based protein production systems to produce Acidothermus cellulolyticus endo-1, 4-?-glucanase (E1) in non-transgenic plants.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.