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 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.
- Research Organization:
- Argonne National Laboratory (ANL)
- Sponsoring Organization:
- NIH
- DOE Contract Number:
- AC02-06CH11357
- OSTI ID:
- 949378
- Report Number(s):
- ANL/ER/JA-40417
- Journal Information:
- Protein Expr. Purif., Journal Name: Protein Expr. Purif. Journal Issue: 1 ; Jun. 2002 Vol. 25
- Country of Publication:
- United States
- Language:
- ENGLISH
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