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Figure 3 | BMC Biotechnology

Figure 3

From: Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

Figure 3

SDS-PAGE and Western blot analysis of the proteins produced. (A) Secreted CBHI-GLA purified via strep2 affinity, measured by strep-tactin-AP Western blot (100 ng loaded). The 50 kD band represents the GLA cleaved by KEX2 protease from the CBHI carrier. The larger band remains as a CBHI-GLA fusion. (B) Comparison of a solubilised batch of purified ZERA-GLA (Z-GLA pure) compared to commercial GLA (GLA control) (1 μg of each). The ZERA-GLA protein runs about 10 kD higher than commercial GLA as expected. (C) The same ZERA-GLA batch compared to ZERA-GLA contained in mycelium (Z-GLA Biomass) and extracted directly from filtered mycelia by boiling in protein sample loading buffer (1 μl of each). Detection by anti ZERA Western blot reveals the presence of several lower molecular weight degradation products contained within the cell, still retaining the ZERA peptide. (D) A TEV protease cleavage of GLA from the ZERA peptide (TEV digest) directly using a 10-fold digest buffer dilution of insoluble enriched protein body material (Z-GLA PB), compared to 0.5 μg of commercial GLA and 1 μg of the purified soluble form of ZERA-GLA. 5 μl were loaded for both the digest and starting PB material. The anti GLA Western blot shows that while cleavage is possible from insoluble PBs three lower molecular weight forms of GLA are present, perhaps due to endogenous protease. (E) A four step pellet wash purification of the ZERA-GLA PB material from a 5 μl sample of 1 g fresh weight diluted in 10 ml (PB) showing the soluble components removed from the PB material with a 5 μl sample of tenfold concentration increase (Wash). The PB pellet is then solubilised in the same volume and centrifuged leaving insoluble debris (Pellet) and the soluble ZERA-GLA (Solubilisation) (5 μl for both).

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