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:4/19/2017)... ... ... A number of new instruments have recently emerged to accommodate different applications ... and Cell Analysis Education Webinar Series , will focus on advances in the Invitrogen™ ... applications. , Many flow cytometers have unique capabilities and the Attune NxT Flow ...
(Date:4/19/2017)... (PRWEB) , ... April 18, 2017 , ... ... Halo Labs . The move comes after the company changed focus to ... market, our new brand and our new technology,” says CEO Robert Hart. Founders ...
(Date:4/19/2017)... ... April 19, 2017 , ... ThermaGenix, the PCR Improvement Company, ... to several other early achievements at ThermaGenix, including the business formation and licensing ... ThermaGenix will use proceeds from the Series A-1 round to:, ...
(Date:4/19/2017)... ... April 19, 2017 , ... ... which makes educational webinars accessible to novices as well as experienced users, attendees ... of commonly performed coagulation screening tests. , Hemostasis testing quality is determined ...
Breaking Biology Technology:
(Date:4/11/2017)... , April 11, 2017 NXT-ID, Inc. ... technology company, announces the appointment of independent Directors Mr. ... to its Board of Directors, furthering the company,s corporate governance ... Gino ... we look forward to their guidance and benefiting from their ...
(Date:4/4/2017)...   EyeLock LLC , a leader of iris-based ... Patent and Trademark Office (USPTO) has issued U.S. Patent ... an iris image with a face image acquired in ... 45 th issued patent. "The ... the multi-modal biometric capabilities that have recently come to ...
(Date:3/29/2017)...  higi, the health IT company that operates the ... , today announced a Series B investment from ... The new investment and acquisition accelerates higi,s strategy to ... population health activities through the collection and workflow integration ... collects and secures data today on behalf of over ...
Breaking Biology News(10 mins):