Evaluación y análisis de riesgos

The lytic activity WT PLY mutants were analysed on the red cell assay plate. The ∆6 PLY mutants were assessed for red blood cell aggregation on a

hemagglutination assay.

4.6.1 Lytic activity of the mutant proteins

All 8 random mutants created in WT PLY background were assessed for their lytic activity. Lytic activity of each of individual mutant is plotted on the graphs (Figure 4.14).

These experiments were set up to identify any other residues apart for the two residues (∆146A∆147R) identified by (Kirkham et al., 2006a) that may be

In WT PLY background; four mutants (1, 6, 19 and 21), the 5 amino acid

insertions had gone into the domain 4 whereas the other four mutants (2, 9, 12 and 13), the insertions had gone into the domains 1-3 of the protein. All the 5 amino insertion mutants in domain 4 were plotted in the same graph (Figure 4.14A) and rest of the mutants were plotted in the other graph (Figure 4.14B).

The lytic activity of these 5 amino acid random mutants for erythrocytes was assessed by indirect measurement of released haemoglobin. The specific

haemolytic activities of the toxins were then calculated in HU/mg and are stated in table 4.3.

Figure 4-14 Haemolytic activity of the mutant proteins

% lytic activity of individual mutant (Y-axis) plotted on the graphs against log toxin concentration (X-axis) of each mutant.

Figure 4.14A showing WT PLY mutants with 5 amino acid insertions in D4 of the toxin resulted in a decrease in lytic activity of the all of them apart from Mutant 6, where the lytic activity is almost comparable to the WT PLY. The 5 amino acid

(a.a) insertions in Mutant 6 WT PLY were exactly at the same place (after 389 amino acids) where the five a.a insertions in pET33bD4PLY had gone (Table 4-1). In case of D4PLY, this 5 amino acid random insertions made the mutant insoluble but in case of full length PLY it was soluble. The 5 amino acids residues inserted in case of Mutant 6 were (CGRRG) where as in D4 background it was (SAAAV) which may have made former soluble and the latter insoluble.

The 5 amino acid insertions in three other mutants in D4PLY have all gone in important positions. In Mutant 21, the 5 amino acid insertion has gone in the undecapeptide region after first tryptophan residues and has made this mutant non-lytic. In Mutant 19, the 5 amino acid insertion has gone very near to the undecapeptide region exactly after 4 amino acids after the undecapeptide region and in Mutant 1; the 5 residues were inserted after the 3rd hydrophobic loop (L-3) of domain 4 PLY (Figure 4.16A).

The results of this study were comparable with the findings of the previous studies (Korchev et al., 1998, Berry et al., 1995) which showed that

undecapeptide region (residues 427-437) is an important region of the toxin and mutation made in this region at one of the first three tryptophan residue 433 (W>F) drastically reduced the lytic activity of the toxin to 1%. Although in this study single amino acids were not substituted, but 5 amino acids were inserted at the same position.

Table 4-3 Specific haemolytic activity and some additional information of the mutant proteins

The lytic activity of the other mutants where the 5 amino acid insertions were in the domains 1, 2 and 3 (Mutant 2, 9, 12 and 13) were similar to the WT PLY suggesting that addition of 5 amino acid residues in other domains does not have an effect on the lytic activity of the toxin. These random mutants had not gone in any well studied regions of the protein. None of these mutants had gone near the two residues (∆146A∆147R) which are responsible for abolishing the lytic activity of the toxin (Figure 4.16A).

4.6.2 Hemagglutination activity of the mutant proteins

The random mutants generated in the ∆6 PLY background were checked for their ability to aggregate/agglutinate red blood cells. In total 9 mutants were

generated; four mutants (Mutant 27.10, 35, 44.1 and 44.2) had 5 amino acids residues inserted in domain 4 and five mutants (Mutant 1, 5, 13, 29.4 and 29.5) had insertions in domains 1, 2 and 3 of the toxin. The assay was done according to the same protocol as mentioned in the materials and methods section 2.9.3.

The tagfree was also causing the same aggregation/ agglutination phenotype as His-tagged purified ∆6 PLY. ∆6 PLY is non-lytic so the specific lytic activity for

these mutants was not calculated and they were only screened for their aggregation activity.

Figure 4-15 Hemagglutination assay of the mutant proteins

1µg of mutant proteins was added in the first well the plate and serial dilutions were made (1:2) across the 96 well plate. The plates were observed for the agglutination of the red blood cells as seen with the ∆6 PLY. 1ng of WT PLY and 50µl PBS were used as positive and negative controls. The small arrows showing the Histagged and tagfree version of ∆6 PLY.

Figure 4-15 showing the different phenotypes as explained in figure 2.5. PLY used as positive control was causing lysis and thus the pinkish tinge observed in the wells. PBS was not causing any effect and hence a red blood cell pellet was seen. The result shows that both the His-tagged and the tagfree PLY (black arrows) were showing aggregation phenotype. Therefore the His-tag seems not to be causing this aggregation phenotype. Mutants 1, 5, 13, 29.4, 29.5 was causing the same aggregation phenotype as cell lysates of the DNA 2.0 Library and Mutants 27.10, 35, 44.1 and 44.2 showing the same aggregation negative phenotype and clearing well effect as seen with TL mutants in figure 3.8.

The results suggest that Mutants 27.10, 35, 44.1 and 44.2, where 5 residues were inserted in the domain 4 of the toxin were aggregation negative and Mutants 1, 5, 13, 29.4 and 29.5 where amino acids were inserted in domains 1, 2 and 3 were agglutination/aggregation positive.

In these hemagglutination assays the same clearing effect of the wells was observed in Mutant 27.10, Mutant 44.2 and Mutant 35 as seen with the TL mutants. The clearing effect however, was less pronounced in Mutant 44.1. These mutations were mapped on the structure and figure 4.16B showing that all the aggregation negative phenotype mutants were near or in the well studied regions of the toxin whereas the aggregation positive phenotype mutants were in domains 1, 2 and 3 (all mutants highlighted in blue).

Table 4.4 states the aggregation/agglutination type, domains of PLY where these 5 amino acid insertions had gone and any important information of all the

purified mutants generated in ∆6 PLY background.

Table 4-4 Haemagglutination results of the mutant proteins