Figure 6

Effect of overexpression HAC1(S) on the expression level of heterologous proteins. A. Surface display: evaluation of Hac1p expression on the surface expression of heterologous proteins. Four proteins were evaluated: mouse interferon-γ (mIFNγ), human interferon-β (hIFNβ), human thrombomodulin (hTM) and human erythropoietin (hEPO). Yellow lines represent the non-displaying strains, purple the strain displaying the surface protein alone, yellow the strains transformed with inducible Hac1p and red the strains displaying the Hac1p constitutively. B. Secreted proteins, mIL-10: Comparison of mIL-10 production of a constitutive Hac1p expressing, inducible Hac1p expressing and the reference strain. The amount of IL-10 protein in culture supernatant was measured by ELISA. Coomassie gel of IL-10 production reference strain versus an inducible Hac1p expressing strain. Lane 2 and 3 illustrate the production of IL-10 protein (arrowheads) in the reference strain before and after endoH treatment respectively. Lane 4 and 5 show the production of the Hac1p overexpressing strain before and after endoH treatment. Kar2p is highly secreted (arrowheads) in the Hac1p expressing strain. Hyperglycosylation is highlighted in boxes. C. Secreted proteins, transialidase: Enzymatic hydrolysis of MUNANA to methyl-umbelliferon in function of time by trans-sialidase present in the medium. Enzymatic release of methyl-umbelliferon was determined every 5 min. D. Membrane proteins: Overexpression of Hac1p leads to a more homogenous dispersed Adenosine A2 receptor (M = monomer, D = dimer). Hac1p overexpression promotes a better processing of the α-mating factor. Radioligand binding studies on membranes of P. pastoris cells expressing the Adenosine A2 receptor with (broken line) and without (solid line) Hac1p.