Paper I

CHiC, a new tandem affinity tag for the protein purification toolbox

During cell division in S. pneumoniae the septal cross wall, which separates the two daughter cells, must be split down the middle by one or more peptidoglycan hydrolases. It is of great interest to identify the key player(s) in this process. It might also be of practical importance, as the enzyme(s) involved can be a valuable drug target. Over the years, several peptidoglycan hydrolases has been suggested to contribute to daughter cell separation. Only LytB has a role in the process, as it is required for dispersal of pneumococcal chains [58]. In recent years, the putative murein hydrolase PcsB has emerged as the leading candidate for the major cross wall splitting enzyme in S.

pneumoniae. However, attempts to demonstrate that PcsB is a peptidoglycan hydrolase have so far failed [57, 94]. Before the present study was started, it was known that PcsB interacts with and probably is controlled by the transmembrane FtsEX complex [57, 62]. This could mean that PcsB must be activated by FtsEX in vivo, and that purified recombinant PcsB is inactive because it adopts the “wrong” conformation in vitro. Another possibility was that the lack of detectable enzymatic activity was due to other factors such as the purification procedure or the assay conditions used. To address these questions, it was decided to express PcsB in E. coli and purify it to near homogeneity. This would allow us to characterize the properties of PcsB, and test its activity under different assay conditions. Another important goal was to produce sufficient amounts of PcsB for crystallization studies and structure determination. For this purpose we needed a procedure that would make it possible to purify PcsB in milligram quantities. Purification of PcsB has previously been shown to be challenging due to the fact that overexpressed native PcsB ends up in inclusion bodies which are difficult to refold [87, 94]. With this in mind, we chose a different approach. We designed a tandem affinity tag based on the choline-binding domain of CbpD. It has been shown previously that pneumococcal proteins with choline-binding domains bind strongly to the diethylaminoethanol groups of DEAE cellulose, and that such domains can be used as an affinity tag when fused to other proteins [77, 78]. In the original description of the system, the choline-binding domain of LytA, which contains six choline-binding sites [76] was

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used. The choline-binding domain of CbpD is shorter and contains fewer choline-binding sites.

The reason I chose the choline-binding domain from CbpD instead of the corresponding domain from LytA was that it is smaller and therefore might interfere less with the folding of the fusion partner. Besides, I knew it worked well, because other members of the research group had already used it as a “natural” affinity tag for the purification of CbpD itself.

In addition to the choline-binding domain of CbpD, the new 16.5 kDa affinity tag termed CHiC was constructed with an N-terminal conventional 6xHis-tag and a proteolytic site specific for the TEV (tobacco etch virus) endopeptidase at the C-terminus. The purposes of the 6xHis-tag are to both be able to perform immobilized metal affinity chromatography (IMAC) before treatment with the TEV protease, and to facilitate the removal of the CHiC-tag after digesting the affinity-purified fusion protein with His-tagged TEV protease (AcTEV^TM protease, Invitrogen). By using IMAC, undigested fusion protein, free CHiC-tag and AcTEV^TM protease can be separated from the purified target protein in a single step.

For the expression of PcsB and the extracellular domain (ECL1) of FtsX, two pRSET A (Invitrogen) based plasmids, pGS01 (PcsB) and pGS02 (ECL1), were constructed. In both cases the CHiC-tag was inserted at the N-terminal end of the target protein. Expression of the fusion proteins, which is driven by the lacT7 promoter, was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). After the first purification step on DEAE cellulose, one liter of cell culture yielded 5-8 mg of CHiC-PcsB and CHiC-ECL1 with a purity of approximately 90-95%.

Further purification by IMAC resulted in more than 95% pure protein, with negligible loss of protein during the purification process. The successful use of the CHiC-tag for purification of PcsB and ECL1 demonstrates that it has the potential to be a valuable new tool for affinity purification of recombinant proteins.

A major Achilles’ heel of the fusion approach is that the affinity tag in most cases has to be removed after purification of the fusion protein. Although highly specific endopeptidases, such as the TEVprotease, have solved the problem of nonspecific cleavage, incomplete processing is still a large problem. Processing efficiency will vary from one fusion protein to another, but in most cases a fraction will remain resistant to digestion. This undigested fraction is easy to remove when using the CHiC-tag system in combination with a His-tagged TEV protease. Another advantage of using the CHiC-tag is that highly purified target protein is obtained in just a few steps, minimizing

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loss of material and manipulations that can result in denaturation of the protein. Furthermore, since large amounts of CHiC-PcsB was obtained in the soluble protein fraction, while previous attempts to purify PcsB mainly gave rise to inclusion bodies, the CHiC-tag has enhanced the solubility of PcsB. Regardless of their origin, more than half of all recombinant proteins overexpressed in E.

coli accumulate in the form of insoluble inclusion bodies [166]. Thus, if the CHiC-tag functions as a general solubility enhancer, by inhibiting aggregation and/or promoting proper folding of its fusion partner, it will help mitigate a major obstacle in recombinant protein production.

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