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 re 3) and enzyme activity assays of the individual fractions after each of the chromatographic steps. The table contains the essential purification parameters.


Data courtesy of Dr Richard Lukacin, Institute for Pharmaceutical Biology, University of Marburg, Germany

Coomassie is a trademark of ICI.


back to top
'"/>

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:
*Name:
*Comment:
*Email:


(Date:10/10/2017)... firm Parks Associates announced today that Tom Kerber , ... Meeting , October 11 in Scottsdale, Arizona . Kerber ... smart safety and security products impact the competitive landscape. ... Parks Associates: Smart Home Devices: Main Purchase Driver ... "The residential security market has experienced continued growth, and the introduction ...
(Date:10/9/2017)... 2017  BioTech Holdings announced today identification and ... ProCell stem cell therapy prevents limb loss in ... demonstrated that treatment with ProCell resulted in more ... compared to standard bone marrow stem cell administration.  ... reduction of therapeutic effect.  ...
(Date:10/9/2017)... ... October 09, 2017 , ... ... October 5, 2017, in the medical journal, Epilepsia, Brain Sentinel’s SPEAC® System ... standard, video EEG, in detecting generalized tonic-clonic seizures (GTCS) using surface electromyography ...
(Date:10/6/2017)... ... October 06, 2017 , ... The ... and technology sector at their fourth annual Conference where founders, investors, innovative practitioners ... and the ELEVATE pitch competition showcasing early stage digital health and med tech ...
Breaking Biology Technology:
(Date:4/18/2017)... 18, 2017  Socionext Inc., a global expert in SoC-based imaging ... server, the M820, which features the company,s hybrid codec technology. A ... Tera Probe, Inc., will be showcased during the upcoming Medtec Japan ... at the Las Vegas Convention Center April ... Click here for ...
(Date:4/13/2017)... Calif. , April 13, 2017 UBM,s ... York will feature emerging and evolving technology ... Both Innovation Summits will run alongside the expo portion ... speaker sessions, panels and demonstrations focused on trending topics ... largest advanced design and manufacturing event will take place ...
(Date:4/11/2017)... BEACH GARDENS, Fla. , April 11, 2017 ... identity management and secure authentication solutions, today announced ... contract by Intelligence Advanced Research Projects Activity (IARPA) ... for IARPA,s Thor program. "Innovation has ... onset and IARPA,s Thor program will allow us ...
Breaking Biology News(10 mins):