Example 3

Recombinant Protein Purification

FIG. 5 shows the steps of one of the purifications carried out on the chimera. In the case of GRNLY, this process was shown in an earlier paper [Ibáñez, R., University of Zaragoza. 2015]. It can be seen in FIG. 5A that the P. Pastoris supernatant obtained after induction (lane 1) contains rather diluted proteins. After concentrating same with Pellicom, protein bands are not seen in the permeate (lane 3), but proteins that are much more concentrated than in the supernatant are seen in the concentrate (lane 2). After dialysis (lane 4), the band profile remains similar to the concentrate. Furthermore, protein bands are not seen in the buffer in which the dialysis bag (lane 5) was introduced. Upon addition of the nickel resin, the chimera binds to said resin as it has a histidine tag. After adding the resin (lane 6), the intensity of a band corresponding to a protein of about 40 kDa decreases with respect to the concentrate and dialysate. This band may correspond to the chimera. The fact that this band does not altogether disappear may indicate that the nickel resin was saturated. In the washes performed on the resin, particularly in the first wash (lane 7), it can be seen how the residues of other proteins are removed. Finally, after the elution of the nickel column, a major protein with a molecular weight of about 40 kDa corresponding to the molecular weight of the chimera (lane 11) is clearly observed. As shown in FIG. 5B, it was confirmed by means of immunoblot that this band of about 40 kDa corresponds to the chimera (lane 11). It is also confirmed that the resin was saturated because a band appears in the post-resin dialysis phase (lane 6).

FIG. 6 shows different elution fractions and the pooling of all of them with the exception of elution fraction 1. FIG. 6A shows several bands in the different elution fractions and in the total eluate. The band with the highest intensity has a molecular weight corresponding to the chimera. Furthermore, other bands having intermediate molecular weights are observed, which means that the chimera undergoes partial proteolysis. The band with the second highest intensity has a molecular weight of about 10 kDa, which corresponds to 9-kDa GRNLY, as its molecular weight increases since it is bound to a histidine tag. In FIG. 6B, it was confirmed by means of immunoblot that these bands of about 40 and 10 kDa correspond to the chimeric recombinant protein and to recombinant GRNLY, respectively.

Once the chimera is generated, its functionality must be assured, that is, on one hand the scFv still recognizes the CEA antigen, and on the other hand GRNLY is still cytotoxic.

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