Short CommunicationConstruction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli
Introduction
The synthesis of the first biologically functional bacterial plasmid (Cohen et al., 1973) signaled the beginning of molecular cloning and recombinant DNA technology. With the advent of recombinant DNA technology, researchers were first able to selectively increase the expression of single genes, which in turn increased yields of protein purifications. However, purification of proteins in their native form is still a difficult and time-consuming process.
Not until the advent of affinity tag purification did protein purification become a faster and more efficient process. Development of affinity tags such as the maltose-binding tag (di Guan et al., 1988, Maina et al., 1988), the polyhistidine tags of the pET vector series, (Rosenberg et al., 1987, Studier and Moffatt, 1986, Studier et al., 1990), and the IMPACT® intein chitin-binding tag (Chong et al., 1997, Chong et al., 1998) allowed for more efficient and rapid purification of diverse proteins at yields much higher than would have been possible through native purification. The development of instruments such as the Maxwell™ 16 Instrument and the Maxwell™ 16 Polyhistidine Protein Purification Kit (Promega), have taken protein purification a step further by now allowing for the automated purification of up to 16 polyhistidine tagged proteins in less than one hour.
While the addition of affinity tags allow for ease of purification, it does render the protein into a non-native state and the affinity tag can often hamper subsequent work with the protein. While the IMPACT® system requires cleavage of the protein from the tag as an elution step, other systems require a subsequent cleavage and purification to remove the affinity tag. One method of tag removal is through the use of the tobacco etch virus (TEV) protease. TEV protease has become one of the proteases of choice for cleaving fusion proteins due to its high degree of specificity, its resistance to many protease inhibitors used in protein purification, and the ease of separation of both the protease and affinity tag from the protein of interest (Parks et al., 1994). Additionally, improvement to the ability to purify large amounts of TEV protease (Blommel and Fox, 2007, Lucast et al., 2001, van den Berg et al., 2006) can make the TEV protease a relatively inexpensive option for cleavage of fusion proteins when compared to other commercially available options.
Previous work has established that various protein fusion vectors featuring rTEV protease cleavage sites are viable for the purification of high levels of recombinant protein through high-throughput approaches (Dummler et al., 2005, Korf et al., 2005) or via ligation independent cloning (Cabrita et al., 2006). Here, we report on the construction of two new series of vectors to the growing family of rTEV-cleavable protein fusion vectors that allow for efficient purification of recombinant bacterial proteins. The pTEV series is based off the Novagen pET vector series and incorporates a TEV protease cleavage site as well as an improved variety of restriction endonucleases sites to increase cloning options. The pKLD series utilizes the pET vector backbone while incorporating both a polyhistidine tag as well as the maltose-binding tag from the New England Biolabs pMAL vector series. The pKLD series also have a TEV protease cleavage site and improved multiple cloning sites.
Section snippets
pTEV plasmids direct the synthesis of proteins fused to a hexahistidine (H6) N-terminal tag
pTEV vectors used to overproduce proteins with TEV-cleavable N-terminal His6 tags were constructed by modifying the Novagen ketosteroid isomerase (KSI) fusion plasmid pET-31b(+). The KSI coding sequence was removed by digestion restriction enzymes NdeI and Bpu1102I(EspI). A hexahistidine (His6) tag coding sequence, a seven amino acid spacer sequence, and a SpeI site were introduced at the NdeI and Bpu1102I sites with the rTEVLink1 DNA fragment (Table 1, supplemental material). This temporary
Acknowledgment
This work was supported by PHS grant GM62203 to J.C.E.-S, and by grant AR35186 to I.R.
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