Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Wu, Z.
Right arrow Articles by Weber, P. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, Z.
Right arrow Articles by Weber, P. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 12, No. 4, 341-348, April 1999
© 1999 Oxford University Press

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase ß-subunit C-terminal mutants

Zhen Wu1, Mark Demma, Corey L. Strickland, Rosalinda Syto, Hung V. Le, William T. Windsor and Patricia C. Weber

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three ß-subunit C-terminal truncation mutants, {Delta}5, {Delta}10 and {Delta}14, which were designed to eliminate a lattice interaction between the ß-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2–3-fold. A new crystalline form of FPT was obtained for the {Delta}10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and {alpha}-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 Å resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.

Keywords: ß-subunit C-terminal mutants/crystallization/high-level expression/protein farnesyltransferase/SPA assay

1 To whom correspondence should be addressed. E-mail: zhen.wu{at}spcorp.com


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
W. Wang, W. W. Prosise, J. Chen, S. S. Taremi, H. V. Le, V. Madison, X. Cui, A. Thomas, K.-C. Cheng, and C. A. Lesburg
Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability
Protein Eng. Des. Sel., July 1, 2008; 21(7): 425 - 433.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
S. Kimura, T. Umemura, and T. Iyanagi
Two-Cistronic Expression Plasmids for High-Level Gene Expression in Escherichia coli Preventing Translational Initiation Inhibition Caused by the Intramolecular Local Secondary Structure of mRNA
J. Biochem., April 1, 2005; 137(4): 523 - 533.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. S. Pickett, K. E. Bowers, and C. A. Fierke
Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate {beta}352 as a Magnesium Ligand
J. Biol. Chem., December 19, 2003; 278(51): 51243 - 51250.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.