Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60076658%3A12310%2F20%3A43901343" target="_blank" >RIV/60076658:12310/20:43901343 - isvavai.cz</a>

Alternative codes found

RIV/61388971:_____/20:00534703

Result on the web

<a href="https://bio-protocol.org/e3616" target="_blank" >https://bio-protocol.org/e3616</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.21769/BioProtoc.3616" target="_blank" >10.21769/BioProtoc.3616</a>

Result language

angličtina

Original language name

Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography

Original language description

Exploring the structure and function of protein complexes requires their isolation in the native state-a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.

Czech name

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Czech description

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Type

J_ost - Miscellaneous article in a specialist periodical

CEP classification

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OECD FORD branch

10608 - Biochemistry and molecular biology

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2020

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Bio-protocol

ISSN

2331-8325

e-ISSN

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Volume of the periodical

10

Issue of the periodical within the volume

10

Country of publishing house

US - UNITED STATES

Number of pages

15

Pages from-to

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UT code for WoS article

000583103500001

EID of the result in the Scopus database

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