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  <I>In Vitro</I> Assembly, Purification, and Crystallization of the Rab Geranylgeranyl Transferase:Substrate Complex

Rak, A., Niculae, A., Kalinin, A., Thomä, N. H., Sidorovitch, V., Goody, R. S., et al. (2002). <I>In Vitro</I> Assembly, Purification, and Crystallization of the Rab Geranylgeranyl Transferase:Substrate Complex. Protein Expression and Purification, 25(1): 1, pp. 23-30. Retrieved from http://dx.doi.org/10.1006/prep.2001.1605.

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Genre: Journal Article
Alternative Title : Protein Expr. Purif.

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 Creators:
Rak, Alexey1, Author           
Niculae, Anca2, Author
Kalinin, Alexandr, Author
Thomä, Nicolas H.2, Author
Sidorovitch, Vadim2, Author
Goody, Roger S.1, Author           
Alexandrov, Kirill1, Author           
Affiliations:
1Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753289              
2Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753286              

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 Abstract: Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alp heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 Angstrom on an in-house X-ray source. (C) 2002 Elsevier Science (USA)

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Language(s): eng - English
 Dates: 2002-06
 Publication Status: Issued
 Pages: -
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 18804
URI: http://dx.doi.org/10.1006/prep.2001.1605
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Title: Protein Expression and Purification
  Alternative Title : Protein Expr. Purif.
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 25 (1) Sequence Number: 1 Start / End Page: 23 - 30 Identifier: -