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Fig. 1 | Microbial Cell Factories

Fig. 1

From: Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris

Fig. 1

a Schematic structure of K28-mCherryHDEL. The K28 chimera contains an N-terminal signal peptide derived from the α-mating factor which is removed by signal peptidase (SP) cleavage after ER import. In the cis-Golgi, endopeptidase Kex2p cleavage removes the pro- and γ-sequence which leads to the separation of α and β. Both subunits remain connected by a single disulphide which was previously formed in the ER. Carboxypeptidase Kex1p removes the C-terminal arginine residue in a trans-Golgi compartment and, thereby, unmasks the HDEL motif. b Western blot analysis of secreted K28-mCherryHDEL from P. pastoris. Cell-free culture supernatants of the recombinant strain GS115 [pPIC9 K28-mCherryHDEL] and the negative control strain GS115 [pPIC9] grown for 120 h in BMM and ×125 concentrated by centrifugal concentrators were separated under non-reducing (left) and reducing (right) conditions by SDS-PAGE and probed with an anti-DsRed antibody. Position and size of the correctly processed toxin is indicated. c Detection of fluorescent K28-mCherryHDEL. GS115 [pPIC9 K28-mCherryHDEL] was grown for 120 h in BMM medium (pH 7) in the presence of protease inhibitors. SDS-PAGE of the ×125 concentrated cell-free culture supernatant under non-reducing conditions shows the pink fluorescent protein and its fluorescence under UV illumination

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