A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site

To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of protei...

Full description

Saved in:
Bibliographic Details
Published in:Protein expression and purification 2002-06, Vol.25 (1), p.8-15
Main Authors: Stols, Lucy, Gu, Minyi, Dieckman, Lynda, Raffen, Rosemarie, Collart, Frank R., Donnelly, Mark I.
Format: Article
Language:English
Subjects:
affinity purification
Amino Acid Sequence
Base Sequence
Binding Sites
Cloning, Molecular
Endopeptidases - genetics
Genetic Vectors
high throughput
ligation-independent cloning
Molecular Sequence Data
Plasmids - metabolism
Protein Binding
Protein Structure, Tertiary
structural genomics
TEV protease
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-independent cloning (LIC) is well suited to robotic cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2001.1603