pPuzzle-eGFP

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	Plasmids were linearized within the genome integration region prior to electroporation (2 kV, 4 ms, GenePulser, BioRad) into electrocompetent P. pastoris. Multicopy integration of HSA expressing clones was done as described by Marx et al. [19] and selected at higher Zeocin concentrations (up to 1000 μg mL-1).

Protocol tips
Plasmids were linearized within the genome integration region prior to electroporation (2 kV, 4 ms, GenePulser, BioRad) into electrocompetent P. pastoris. Multicopy integration of HSA expressing clones was done as described by Marx et al. [19] and selected at higher Zeocin concentrations (up to 1000 μg mL-1).

Publication protocol

"Cloning and transformation was done using the in-house vector pPuzzle [15], which contains a Zeocin resistance cassette for selection in both E. coli and yeast, an expression cassette for the gene of interest (GOI) consisting of a multiple cloning site and the S. cerevisiae CYC1 transcription terminator, and a locus for integration into the P. pastoris genome (3´ AOX1 region or rDNA locus). Promoter sequences (up to 1000 bps upstream of the start codon of their respective genes) were PCR-amplified from P. pastoris genomic DNA (primer sequences see Additional file 1: Table S1). The promoters were ligated into pPuzzle in front of the start codons of the model proteins, using the ApaI and the SbfI restriction sites of the multiple cloning site of the vector. Vectors expressing the respective model protein under control of PGAP were used as controls throughout the study. For the expression of heterodimeric HyHEL antibody Fab fragment (HyHEL Fab), the expression cassettes of light chain and Fab heavy chain (each under control of PG1) were combined into one vector (using the strategy described in [27]).

HSA was secreted by its native secretion leader, while for CpB and HyHEL Fab the S. cerevisiae alpha mating factor signal sequence was used. To avoid positional effects on reporter gene expression levels, genome integration of the expression plasmids was targeted to either the 3´flanking region of the AOX1 gene or the ribosomal DNA locus (rDNA, for multicopy integration) of P. pastoris, respectively.

Plasmids were linearized within the genome integration region prior to electroporation (2 kV, 4 ms, GenePulser, BioRad) into electrocompetent P. pastoris. Multicopy integration of HSA expressing clones was done as described by Marx et al. [19] and selected at higher Zeocin concentrations (up to 1000 μg mL-1).

P. pastoris cells were first selected and cultivated in petri dishes on YPD agar and then inoculated in an YPG medium as pre-culture for screenings and fermentations. Antibiotic selection by Zeocin was applied on plates and in pre-culture at a concentration of 25 μg mL-1 or higher."

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