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.
Stols
Lucy
L
Environmental Research Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Gu
Minyi
M
Dieckman
Lynda
L
Raffen
Rosemarie
R
Collart
Frank R
FR
Donnelly
Mark I
MI
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D010450:Endopeptidases; C099488:TEV protease