P URIFICATION OF RECOMBINANT ANTIGENS FROM E. COLI

The antigens selected (section 3.1) were cloned and expressed in salmonella carrier strains. The ability of the candidate antigens to induce a protective immune response in animal models was determined using these strains. Due to their novelty, immunological reagents like specific antibodies or purified protein were unavailable.

Antigen Surface Cytosol

Therefore it was necessary to express and purify those antigens, to enable analyses of the immune response like detection of specific antibodies from serum, re-stimulation of antigen-specific T cells isolated from vaccinated individuals or for better characterization of the antigens, generation of specific polyclonal antibodies. The following section describes the antigen purification process, i.e. the generation of expression plasmids, the induction of recombinant proteins and their purification.

Fig. 3.10: Overview of cloning of LinJ08.1140 into pET28(+) expression plasmids Shown are plasmid maps (A) before and after insertion of LinJ08.1140 gene into the expression plasmids. Below the maps the cloning site sequence is depicted (B), with promoter, operator, RBS, His-tags, gene and terminator sequences marked. Note: due to the stop codon in the gene of interest the second His-tag was lost.

To avoid the time-consuming process of determining purification strategies based on physical properties which may differ for each antigen, it was decided to add to antigens a hexahistidine tag which allowed purification using nickel affinity columns.

For this, antigens were excised from the cytosolic expression plasmids (see section 3.2.3) using NdeI and BamHI restriction enzymes, ligated into another expression plasmid pET28(+) (Novagen) which provided an N-terminal His-tag (fig. 3.10) and subsequently transformed into E. coli XL-1. All constructs were confirmed by PCR, restriction digestion and sequencing.

For antigen expression, these constructs were transformed into E. coli strain BL21 CodonPlus (DE3)-RIPL (Stratagene), in which induction of expression by IPTG was tested on three colonies. Positive colonies showing strong protein expression were then selected to prepare bacterial stocks. All subsequent protein induction experiments were performed using these stocks. Figure 3.11 shows SDS PAGE gels of bacterial lysates prepared before and after the addition of IPTG.

Fig. 3.11: IPTG induced expression of leishmania proteins

Recombinant protein expression was induced by addition 0.4 mM IPTG and samples were taken before (-) and after (+) induction. Samples were boiled in protein sample buffer and applied onto either 12 % SDS-gels (LinJ09.1180, LinJ25.1680) or 15 % SDS-gels (KMP-11, LinJ08.1140, LinJ23.0410, LinJ35.0240)

Titration experiments showed that most of the antigens were inducibly expressed but this required up to 1mM of IPTG. However, LinJ35.0240 antigen did not show any expression induction even at this concentration of IPTG. Furthermore, incubation at lower temperatures (22 ºC) or variation of IPTG concentration did not lead to any detectable protein expression (data not shown).

Purification of the His-tagged antigen using FPLC under native conditions was only successful for KMP-11 (see figure S-3 in Supplementary material), which was then dialysed against PBS to remove imidazole and concentrated by ultrafiltration.

Fig. 3.12: Purification of recombinant leishmania antigens from inclusion bodies Shown is the chromatogram of the purification of LinJ08.1140 from inclusion bodies.

Fractions were collected and those representing the first peak (grey box) were applied on an SDS gel. Fractions with higher protein content were pooled and further

processed. FT: flow through

The amount of protein was determined and aliquots were frozen at -80 ºC. All other antigens did not show an absorption peak during elution and consequently no positive fractions were detected by Coomassie staining of SDS PAGE gels despite being highly inducible with IPTG. It was concluded that overexpression of LinJ08.1140, LinJ09.1180, LinJ23.0410 and LinJ25.1680 resulted in the formation of inclusion bodies. After lysis, treatment with ultrasound did not dissolve the protein sample as

not shown). Alterations in growth conditions, e.g. less IPTG, different induction times and bacterial density did not prevent the formation of inclusions (data not shown).

Therefore it was decided to purify the inclusion bodies and dissolve them in urea and guanidine hydrochloride. The dissolved and denatured proteins were applied on a nickel packed affinity column and bound proteins were re-folded on-column. Eluted fractions were tested for the presence of protein by an SDS PAGE and positive fractions were pooled. An example for a successful purification of proteins from inclusion bodies is shown in figure 3.12.

Imidazole has immuno-stimulatory properties and can influence the response of T cells in re-stimulation assays. To avoid this in future experiments, dialysis against TBS was performed. Unfortunately, once imidazole was removed from the solution, most of the proteins aggregated and became insoluble. However, T cell activation is mediated via MHC-peptide/TCR complexes, hence natural conformation of the antigen is not required. Therefore, the denatured protein was stored in 50 % glycerol at -20 ºC and was used for subsequent T cell assays.

In contrast, for the detection of host antibodies against vaccination antigens, it is important to have the antigen in its natural state, since antibodies are also produced against conformational determinants. In this case, imidazole was not expected to influence antigen-antibody interaction, therefore its removal was not necessary and positive fractions from the FPLC were pooled for subsequent ELISA tests. After determination of protein concentration, ELISA plates were coated with 2.5 μg antigen per well; sealed and stored at 4 ºC until further use.