Navigation Links
Purification of Recombinant Flavonone 3beta-Hydroxylase Using Bio-Scale CHT5-1 Hydroxyapatite Column


Introduction
Flavanone 3b-hydroxylase (FHT), a typical 2-oxoglutaratedependent dioxygenase, catalyzes the 3b-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols which are intermediates in the biosynthesis of flavanoids, catechins, proanthocyanidins, and anthocyanidins in plants. To understand the complex reaction mechanism, the active site of the hydroxylase, and especially its interaction with the substrates and cofactors of the enzyme reaction, knowledge of the crystal structure of the protein is required.

The E. coli-expressed enzyme from Petunia hybrida was purified to apparent homogeneity by a three-step chromatographic procedure which included rapid, high resolution chromatography on a 5 ml Bio-Scale CHT5-I ceramic hydroxyapatite column (Bio-Rad, catalog # 751-0023).

Results Recombinant flavanone 3b-hydroxylase was expressed in E. coli. The cells were lysed and the resulting supernatant from a 45% saturated ammonium sulfate precipitation step was fractionated on a butyl HIC column. The FHT-containing fraction was concentrated and further fractionated using a gel filtration column. The FHT-containing fractions from the gel filtration column were pooled and loaded directly onto a 5 ml Bio-Scale CHT5-I column. Bound proteins were eluted by a two-step linear gradient from 2550 mM in 10 ml and 50200 mM potassium phosphate in 40 ml (Figure 1). Pure enzyme was obtained in the fractions containing 5080 mM potassium phosphate. The purity of the enzyme was investigated by polyacrylamide gel electrophoresis (Figure 2).


The purification procedure was monitored by immuno dot blot analysis for FHT protein (Figu
'"/>

Source:


Page: All 1 2

Related biology technology :

1. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Yeast Protein Production System Features High Yields and One-Step Purification
4. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
5. Easy Cosmid DNA Purification with StrataPrep Kits
6. MinElute PCR Purification Kit
7. QIAquick PCR Purification Kit
8. Microarray Target Purification Kit and the Microarray Target Synthesis Kit (T7)
9. Double-Affinity Purification of GST-His Tagged Proteins Using Vivapure 8-to-96 Well Cobalt-Chelate Kit
10. Ultra-Fast Purification of Small Nucleic Acids
11. RNAClean - RNA & cDNA in vitro Reaction Purification
Post Your Comments:
(Date:7/29/2014)... 29, 2014 The advanced optical methods ... that are driving a revolution in the neurosciences were ... journal published by SPIE, the international society for ... with the launch of the BRAIN Initiative in the ... lay out a vision for and provide examples of ...
(Date:7/29/2014)... ultralight foam of atom-thick sheets can be made to ... at Rice University. , In microscopic images, the ... floors and walls that reinforce each other. The structure ... walls of graphene oxide that self-assemble with the assistance ... say the foam could find use in structural components, ...
(Date:7/29/2014)... July 29, 2014 Second generation biofuels are ... by fossil fuels. However, as natural gas is very cheap, ... they are not as valuable as they could be in ... difficulty financing projects, has made it difficult for renewable energy ... get off the ground. For these reasons, certain companies have ...
(Date:7/29/2014)... 29, 2014 A new report ... (types, applications and geography) - Size, Share, Global Trends, ... and Forecast, 2013 - 2020", suggests that the global ... by 2020 registering a CAGR of 11.1% from 2014 ... by the electro surgical devices in operative procedures by ...
Breaking Biology Technology:SPIE Launches 'Neurophotonics' Journal with Special Section on BRAIN Initiative 2SPIE Launches 'Neurophotonics' Journal with Special Section on BRAIN Initiative 3SPIE Launches 'Neurophotonics' Journal with Special Section on BRAIN Initiative 4Tough foam from tiny sheets 2Renewable Waste Intelligence: Could Renewable Chemicals Be the Future for the Renewable Energy Industry? 2Global Surgical Equipment Market is expected to reach $12.1 billion by 2020 - Allied Market Research 2Global Surgical Equipment Market is expected to reach $12.1 billion by 2020 - Allied Market Research 3
... prescription ... solution, ... new Xtremityscan System and give away a free unit at the ... Xtremityscan is a digital shape acquisition system that allows users ...
... to Create One of the Industry,s Largest eClinical ... ... PAREXEL International,Corporation (Nasdaq: PRXL ), a leading global biopharmaceutical ... plc, a company traded on the London Stock Exchange, ("ClinPhone"),have ...
... June 13 On Thursday, June 19, 2008,at 7:00 ... Time,members of Cell Therapeutics, Inc.,s (CTI) (Nasdaq and MTA: ... of the Annual Meeting,of Shareholders at its Seattle headquarters ... meeting will be webcast live on CTI,s website,at ...
Cached Biology Technology:PAL Health Technologies Debuts Xtremityscan System at Western Podiatric Medical Congress 2PAREXEL Reaches Agreement To Acquire ClinPhone 2PAREXEL Reaches Agreement To Acquire ClinPhone 3PAREXEL Reaches Agreement To Acquire ClinPhone 4PAREXEL Reaches Agreement To Acquire ClinPhone 5Cell Therapeutics, Inc. Special Meeting in Lieu of Annual Meeting of Shareholders Scheduled for Thursday, June 19, 2008 2
(Date:7/30/2014)... the first high-resolution map of the carbon stocks ... Per. The new and improved methodology used to ... future market-based carbon economies. The new carbon ... and it provides the critical input to studies ... use, and enforcement purposes. The technique includes the ...
(Date:7/30/2014)... in the journal Scientific Reports , published by ... Ganguly found that while global temperature is indeed increasing, ... instance, while each year,s average hottest and coldest temperatures ... fall within a wider range of potential high and ... means that even as overall temperatures rise, we may ...
(Date:7/29/2014)... by academics in the University of Bristol,s Nutrition and ... liquid calories into account when planning meals. , The ... Study of Ingestive Behavior Conference (SSIB 2014) in Seattle, ... that we do. , The team was led by ... of Experimental Psychology. , As part of a Biotechnology ...
Breaking Biology News(10 mins):Peru's carbon quantified: Economic and conservation boon 2Peru's carbon quantified: Economic and conservation boon 3Big data confirms climate extremes are here to stay 2
... Fusarium samples. A Virginia Tech plant pathologist ... detect airborne pathogens above agricultural fields. , ... and weed science in the College of Agriculture ... and engineering with his interdisciplinary research but has ...
... common practice among many college students involves "pulling all-nighters", ... practice associated with lower grade-point averages compared to those ... abstract that will be presented Wednesday at SLEEP 2007, ... Societies (APSS). , "Sleep in college students is generally ...
... a microbial protein to improve water quality. , But ... from potentially toxic nanoparticles in their own environments, and ... the same thing on a larger scale. , ... and Lawrence Berkeley National Laboratory found that bacteria from ...
Cached Biology News:Small, self-controlled planes combine plant pathology and engineering 2Small, self-controlled planes combine plant pathology and engineering 3Microbes at work cleaning up the environment 2
...
...
...
... High Resolution - Efficiently ... 50 to 1000 bp. ... sizing PCR fragments, small ... restriction enzyme digestion, and ...
Biology Products: