Figure 4

IGRP toxicity and development of IC-Tagging in bacteria. (a) Coomassie-blue PAGE analysis of extracts from Sf9 cells uninfected (1) or infected with or recombinant baculoviruses directing the expression of IGRP (2), muNS-Mi (3), or co-infected with the muNS-Mi and IC-IGRP baculoviruses (4). The position corresponding approximately to the size of muNS-Mi (21 kDa) is indicated at the left of the picture, as is the 50 kDa marker as a reference for the IC-IGRP approximate size (55 kDa). (b) Extracts from un-induced (1) or IPTG-induced bacteria (2) transformed with an expression plasmid containing the muNS-Mi gene were analyzed by PAGE and stained with Coomassie blue. The right side of the figure shows the supernatant (3) and pellet (4) of protein muNS-Mi purified from a sample similar to the one shown in lane 2 by EDTA treatment (see text). (c) The EM picture shows MS inside bacteria where the expression of muNS-Mi was induced. The arrows indicate the positions of the MS. The black bar represents a distance of 1 µm. (d) IC-tagging working in bacteria. Extracts from un-induced (1) and IPTG-induced (2) bacteria transformed with a dual recombinant plasmid that directs the simultaneous expression of proteins muNS-Mi and HaloTag-IC were analysed by PAGE and Coomassie-blue staining. Lane 3 shows a sample of the partial purification of MS from a sample similar to the one shown in lane 2. The positions corresponding to the calculated size of both proteins are indicated.