Example 1
Lys68 is a globular endolysin, i.e. does not exhibit an apparent domain structure with an enzymatic domain and a cell wall binding domain, as encountered for various other endolysins. The inventor hypothesized, that Lys68 endolysin may nonetheless exhibit a core region responsible for enzymatic activity and tested this hypothesis with truncated versions of Lys68, namely Lys68(1-132) (SEQ ID NO:32), Lys68(1-148) (SEQ ID NO:33) and Lys68(7-162) (SEQ ID NO:34).
Briefly, the following experiment was carried out: Exponentially growing P. aeruginosa cells were harvested by centrifugation and subsequently resuspended in 0.05 M Tris/HCl pH 7.7 buffer saturated with chloroform. This cell suspension was incubated for 45 minutes at room temperature. Afterwards, cells were washed with 20 mM HEPES pH 7.4 and finally adjusted to an OD600 of ca. 1.5 with 20 mM HEPES pH 7.4. In order to test the muralytic activity, 270 μl of chloroform treated cells were mixed with 30 μl of purified variants of Lys68 in a 96 well plate and the OD600 was monitored in a microplate reader.
The result is shown in
