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2.2 Protease-Deficient Strains

Page 90

However, during long bioreactor cultivations, vacuolar proteases, such as proteinase A (Pep4) and B (Prb1), such as proteinase A (PEP4) and B (PRB1) can be released into the culture supernatant through cell lysis, resulting in proteolytic product degradation.

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However, during long bioreactor cultivations, vacuolar proteases, such as proteinase A (Pep4) and B (Prb1), can be released into the culture supernatant through cell lysis resulting in proteolytic product degradation.

2.4 Special Strains

Page 92

Applicability of the ku70 deletion strain was demonstrated for the generation of his4 and ade1 mutants (auxotroph for histidine and adenine, respectively), showing over 90 % HR ef fi ciency with only 250-bp fl anking ends.

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Applicability of the ku70 deletion strain was demonstrated for the generation of his4 and ade1 mutants (auxotroph for histidine and adenine, respectively), showing over 90 % HR efficiency with only 250-bp fl anking ends.

Page 93

Despite the observation of sporulation events that resulting in cells that produce would lead to cells producing alternatively the heavy or the light chain of the antibody, the majority of the population was shown to maintain a diploid state for 240 h of methanol induction, and to secrete the targeted monoclonal antibody to comparable titers and with similar glycan quality compared to a haploid reference strain.

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Despite the observation of sporulation events that result in cells producing alternatively the heavy or the light chain of the antibody, the majority of the population was shown to maintain a diploid state for 240 h of methanol induction, and to secrete the targeted monoclonal antibody to comparable titers and with similar glycan quality compared to a haploid reference strain.

4.2.2 Alternative Inducible Promoters

Page 101

In a systematic approach using DNA microarrays to fi nd promoters that are repressed under glycerol-excess batch conditions, the promoter of a gene coding for a P. pastoris glucose transporter with high affinity (GTH1) was identified and used in a in a methanol-free glucose-limited fed-batch process yielding 1.0 g/L secreted human serum albumin [84].

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In a systematic approach using DNA microarrays to fi nd promoters that are repressed under glycerol-excess batch conditions, the promoter of a gene coding for a P. pastoris glucose transporter with high affinity (GTH1) was identified and used in a methanol-free glucose-limited fed-batch process yielding 1.0 g/L secreted human serum albumin [84].

Table 4 Inducible promoters for recombinant protein production in Pichia pastoris

Page 102

Column: Overexpressed protein

P. lycii phytase

H. brasiliensis HNL

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P. lycii phytase

H. brasiliensis HNL