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Figure 3 | Microbial Cell Factories

Figure 3

From: Application of a wide-range yeast vector (CoMedâ„¢) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts

Figure 3

(A) Physical map of the expression/integration vector pAL-ALEU2m-TEF-MATalpha-IL6 used in this study. The vector contains the 25S rDNA sequence of A. adeninivorans (rDNA, white box), the selection marker ALEU2m (grey segment) and an expression cassette for the IL6 gene in the order A. adeninivorans- derived TEF1 promoter (TEF1 pro, grey segment), the IL6-coding sequence, S. cerevisiae-derived PHO5 terminator(PHO5 ter, black bar) as selection marker. The vector contains a unique Nco I site for linearization within the rDNA sequence. (B) IL6 accumulation in recombinant S. cerevisiae, A. adeninivorans budding cell and mycelia cultures as well as in H. polymorpha. The strains were cultured in YMM supplemented with 2% glucose for 72 h at 30°C (S. cerevisiae, A. adeninivorans budding cells, H. polymorpha) or 45°C (A. adeninivorans mycelia). 20 μl aliquots of culture media were separated on (11%) SDS-PAGE gels, transferred to nitrocellulose filters and probed with anti-IL-6 antibodies. The concentration of recombinant IL-6 was calculated from the signal intensity of a IL-6 standard. (1) IL-6 standard (E. coli), (2) A. adeninivorans G1211/pAL-ALEU2m-TEF-MATα-IL6 – budding cells (205 mg l-1), (3) A. adeninivorans G1211/pAL-ALEU2m-TEF-MATα-IL6 – mycelia (144 mg l-1), (4) S. cerevisiae SEY6210/pAL-ALEU2m-TEF-MATα-IL6 (95 mg l-1), (5) H. polymorpha HP102/pAL-ALEU2m-TEF-MATα-IL6 (90 mg l-1). (C) N-terminus of IL-6 secreted from recombinant S. cerevisiae, A. adeninivorans (budding cell and mycelial cultures) and from H. polymorpha strains.

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Microbial Cell Factories

ISSN: 1475-2859

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