Fig. 7

Gene expression from the nanochromosome supports production of a traditionally integrated extraneous gene, or can be used directly to produce recombinant protein.(a) Expression of PDI^H on nChr 2A could assist formation of the 40 disulfide bonds of mFH encoded by DNA integrated into Chr 4. (b) SDS-PAGE (Coomassie staining) of crude cell-culture media (30 µL, reducing conditions, treated with Endo H_f) before (d0), and three days after (d3), methanol induction. As expected, co-expression of PDI^H located on either nChr 2A or (as a control) a native chromosome, is required for detectable quantities of mFH to be secreted. (c) A Western blot was used to demonstrate PDI^H production in cells containing nChr 2A three days post-induction. (d) A 165-kDa fusion GFP:FH fusion protein is detectable three days after induction in the cell-culture media from two strains containing nChr 2A.2 carrying both PDI^H and GFP:FH. In each lane 40 µL of EndoH_f–treated crude culture medium (after cell removal) was loaded under reducing conditions. Bands were detected with Coomassie blue and by Western blot using an anti-GFP primary antibody that also revealed the presence of a GFP-containing proteolytic fragment. (e) Both mFH and GFP:FH produced herein are cofactors for (30 nM) complement FI-catalysed cleavage of the 110-kDa α’-chain of complement protein C3b into 63-kD and 39-kDa fragments. In each case 12 µL of crude culture medium (at d(ay) 3 or, as a control, at d(ay) 0) was assayed. A potential contribution of non-specific protease activity is excluded by performing control reactions lacking FI.