Reassembly of Synechocystis sp. PCC 6803 F1‐ATPase from its over‐expressed subunits

FEBS Letters

Engelbrecht, S.¹  Steinemann, D.¹  Lill, H.¹ 

[1] Abt. Biophysik, Universität Osnabrück, D 49069 Osnabrück, Germany

关键词: ATPase;    F0F1;    Purification;    Expression;    Synechocystis;    ACMA;    9-amino-6-chloro-2-methoxyacridine;    Chl;    Chlorophyll;    DTT;    dithiothreitol;    EDTA;    ethylenediamine-tetraacetic acid;    GdnHCl;    guanidinium hydrochloride;    Mega 9;    nonanoyl-N-methyl-glucamide;    PCR;    polymerase chain reaction;    PMS;    phenazinemethosulfate;    SDS-PAGE;    sodiumdodecylsulfate-polyacrylamidegelelectrophoresis;    TNP · ATP;    trinitrophenyl-adenosinetriphosphate;    tricine;    N-Tris-(hydroxymethyl) methyl glycine;    Tris;    N-Tris-(hydroxymethyl)-aminomethane;   

DOI  :  10.1016/0014-5793(95)00238-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Subunits α, β, and γ of the F₁-part of cyanobacterial F₀F₁-ATPase have been cloned into expression vectors. Overexpressed subunit β was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant α and γ subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the α and β subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F₁-ATPase was assembled from its purified subunits α, β, γ, δ and ϵ. The reassembled enzyme reconstituted ATP synthesis in F₁-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.

【 授权许可】

Unknown