Ligation independent cloning vectors for expression of SUMO fusions
Section snippets
Enzymes, chemicals, media and strains
Enzymes required for the cloning steps were purchased from New England Biolabs (NEB, Ipswich, MA) unless otherwise stated. All reagents for media were bought from Fisher Scientific (Pittsburgh, PA). All chemicals were purchased from Sigma–Aldrich (St. Louis, MO) unless otherwise stated. PCR primers were purchased from MWG-Biotech Inc. (High Point, NC). For the traditional cloning steps six thymidine residues were included at the 5′ ends of all primers to ensure efficient cleavage by restriction
Vectors
Four LIC vectors have been constructed, derived from the combinatorial pairing of two different parent vector backbones with two different affinity tags (Table 1). The two parent vectors (pET and pASK) use different promoters to drive expression of the cloned fusion protein (Fig. 1a). The two affinity tags are both small peptides and are incorporated upstream of the S. cerevisiae SUMO sequence (Fig. 1b).
The pET vectors allow for the LacI-repressed but strongly inducible production of transcript
Discussion
We have designed a generic approach to recombinant protein production that combines the simplicity of LIC [12] with the strength of the SUMO fusion system [21]. The procedure allows for the systematic cloning of target genes, independent of their sequence, and provides a platform for the standardized purification of recombinant proteins having either native or engineered amino terminal residues. The procedures described here are equally applicable in small laboratory settings focusing on a few
Acknowledgements
The authors thank Kimberly Grasty for technical support. This work was supported in part by NIH grants GM64676 and MH73060 (P.J.L.) and by the Fanny Ripple Foundation.
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