A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.
A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies.
Chanda
Pranab K
PK
Neuroscience Discovery Research, Wyeth Research CN-8000, Princeton, NJ 08543, USA. chandap@wyeth.com
Edris
Wade A
WA
Kennedy
Jeffrey D
JD
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics
D008024:Ligands