A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli
Section snippets
Materials and methods
Plasmids pET-21a(+) and pET-16b(+) were purchased from Novagen. Plasmid containing the dsbG gene was a gift from Dr. Zhai from Peking University at Beijing, China. Plasmid pLG339 was purchased from ATCC, Rockville, MD. E. coli thioredoxin and dsbA genes were obtained from Dr. Edward R. LaVallie from Wyeth Research, Cambridge, MA. Host strains Escherichia coli DH5α and BL21 (DE3) were purchased from Life Technology Limited.
Enzymes were purchased from New England Biolabs, Promega and Life
Construction of LIC-independent expression vector
The LIC-independent expression vectors used in this study are all T7 promoter based expression plasmids. Briefly, a double stranded 92-bp LIC cassette was generated through annealing and ligation of four separate single stranded oligonucleotides as mentioned in Materials and methods. The LIC cassette contains an optimal ribosome binding site, an AUG start codon, a LIC cloning site, and a H6 tag for protein purification. The cassette was flanked by XbaI and XhoI restriction sites at the 5′- and
Discussion
We have described in this paper the development of LIC-independent expression vectors for co-expression of up to three bacterial genes (dsbA, dsbG, and trx) simultaneously in the same E. coli cells as well as the co-expression of two mammalian genes, KChIP1 and Kv4.3. Other Kv channels, as for example, rat voltage-gated Kv1.1 α-subunit was also found to exist as an octomeric structure with its β2-subunit [38]. Multiple subunits association may contribute to channel formation, some serving as
Acknowledgments
We gratefully thank Dr. Zhonghe Zhia from Peking University, Beijing, People’s Republic of China for plasmid containing dsbG, Dr. Edward R. LaVallie from Wyeth Research, Cambridge, MA for dsbA and trx genes and Bart Nieuwenhuijsen for critically reading the manuscript.
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