A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli

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Abstract

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 (6× 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 6× 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.

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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