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

D. Steinemann, Siegfried Engelbrecht, H Lill

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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

Original languageEnglish
Pages (from-to)171-4
Number of pages4
JournalFEBS Letters
Volume362
Issue number2
DOIs
Publication statusPublished - 3 Apr 1995

Fingerprint

Synechocystis
Proton-Translocating ATPases
Adenosine Triphosphate
Thylakoids
Inclusion Bodies
Cell Extracts
Escherichia coli
Nucleotides
Membranes
Enzymes

Keywords

  • Adenosine Triphosphate
  • Binding Sites
  • Cloning, Molecular
  • Cyanobacteria
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression
  • Genetic Vectors
  • Macromolecular Substances
  • Protein Folding
  • Proton-Translocating ATPases
  • Recombinant Proteins
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Steinemann, D. ; Engelbrecht, Siegfried ; Lill, H. / Reassembly of Synechocystis sp. PCC 6803 F1-ATPase from its over-expressed subunits. In: FEBS Letters. 1995 ; Vol. 362, No. 2. pp. 171-4.
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abstract = "Subunits alpha, beta, and gamma of the F1-part of cyanobacterial F0F1-ATPase have been cloned into expression vectors. Over-expressed subunit beta was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant alpha and gamma subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the alpha and beta subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1-ATPase was assembled from its purified subunits alpha, beta, gamma, delta and epsilon. The reassembled enzyme reconstituted ATP synthesis in F1-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.",
keywords = "Adenosine Triphosphate, Binding Sites, Cloning, Molecular, Cyanobacteria, Electrophoresis, Polyacrylamide Gel, Gene Expression, Genetic Vectors, Macromolecular Substances, Protein Folding, Proton-Translocating ATPases, Recombinant Proteins, Journal Article, Research Support, Non-U.S. Gov't",
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Reassembly of Synechocystis sp. PCC 6803 F1-ATPase from its over-expressed subunits. / Steinemann, D.; Engelbrecht, Siegfried; Lill, H.

In: FEBS Letters, Vol. 362, No. 2, 03.04.1995, p. 171-4.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Reassembly of Synechocystis sp. PCC 6803 F1-ATPase from its over-expressed subunits

AU - Steinemann, D.

AU - Engelbrecht, Siegfried

AU - Lill, H

PY - 1995/4/3

Y1 - 1995/4/3

N2 - Subunits alpha, beta, and gamma of the F1-part of cyanobacterial F0F1-ATPase have been cloned into expression vectors. Over-expressed subunit beta was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant alpha and gamma subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the alpha and beta subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1-ATPase was assembled from its purified subunits alpha, beta, gamma, delta and epsilon. The reassembled enzyme reconstituted ATP synthesis in F1-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.

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

KW - Adenosine Triphosphate

KW - Binding Sites

KW - Cloning, Molecular

KW - Cyanobacteria

KW - Electrophoresis, Polyacrylamide Gel

KW - Gene Expression

KW - Genetic Vectors

KW - Macromolecular Substances

KW - Protein Folding

KW - Proton-Translocating ATPases

KW - Recombinant Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/0014-5793(95)00238-5

DO - 10.1016/0014-5793(95)00238-5

M3 - Article

VL - 362

SP - 171

EP - 174

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

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