P. pastoris, GS115 and Escherichia coli, DH5α strains were purchased from Invitrogen (Carlsbad, CA, USA). The pHBM905BDM plasmid was constructed in our laboratory. All culture media, including minimal dextrose (MD,1.34% YNB, 2% dextrose, 4 × 10−5% biotin), buffered minimal glycerol (BMGY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 1% glycerol), and buffered minimal methanol (BMMY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 0.5% methanol) were prepared, as described in the P. pastoris expression manual (Invitrogen).
P pastoris
Manufactured by Thermo Fisher Scientific
Sourced in United States
P. pastoris is a yeast species commonly used in biotechnology as an expression system for recombinant protein production. It is a single-celled eukaryotic organism that can be genetically engineered to express high levels of heterologous proteins.
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Lab products found in correlation
Escherichia coli trans1 t1, by Transgene (2 mentions)
Ppic9, by Thermo Fisher Scientific (2 mentions)
Escherichia coli dh5α, by Thermo Fisher Scientific (1 mentions)
P pastoris expression manual, by Thermo Fisher Scientific (1 mentions)
P pastoris gs115, by Thermo Fisher Scientific (1 mentions)
Peasy t3 vector, by Transgene (1 mentions)
Pichia expression kit, by Thermo Fisher Scientific (1 mentions)
Ni nta, by GE Healthcare (1 mentions)
α mannosidase, by Merck Group (1 mentions)
Quantofix, by Merck Group (1 mentions)
Proteomics grade trypsin, by Promega (1 mentions)
Endo hf, by New England Biolabs (1 mentions)
Ni nta, by Thermo Fisher Scientific (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Bioflo 115, by Eppendorf (1 mentions)
7 protocols using p pastoris
1
Yeast and Bacterial Expression Protocols
2
Heterologous Expression of Tlswo
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Talaromyces leycettanus JCM12802, the donor strain, was purchased from Japan Collection of Microorganisms RIKEN BioResource Center (Tsukuba, Japan). Escherichia coli Trans I-T1 (TransGen, Beijing, China) was used for routine gene cloning. T. reesei AST1116 and P. pastoris GS115 (Invitrogen, Carlsbad, CA, United States) were used as hosts for gene expression. pPIC9 (Invitrogen) and pTrEno plasmids of were used to drive Tlswo gene expression in P. pastoris and in T. reesei, respectively. The pTrEno plasmid was constructed described by Linger et al. (2015) (link).
3
Cloning and Mutagenesis of AtABCG1 in P. pastoris
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As previously described, a synthetic gene of AtABCG1 (At2g39350) with codon optimization for expression in P. pastoris (Invitrogen) was cloned by In-Fusion into the expression vector pSGP18-Ntag41 (link). An ATPase hydrolysis deficient AtABCG1 mutant was generated by site-directed mutagenesis. First Glu259 in the conserved Walker B motif was replaced by Gln with the primer pairs ABCG1-EQ fwd/rev and additionally His291 of the H-loop was replaced by Ala with the primer pairs ABCG1-HA fwd/rev (Table S1 ). Sequences were verified by DNA sequencing (GATC Biotech).
4
Heterologous Expression of Penicillium pg63
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The recombinant plasmid used in this work was pPIC9-pg63, which contained the gene pg63 (HQ446162) from the strain Penicillium sp. CGMCC 166923 . Site-directed mutagenesis was carried out using the specific primers (Additional file 6) and two-step polymerase chain reactions (PCRs). All mutants were verified by double-stranded plasmid sequencing. Escherichia coli Trans I-T1 (TransGen, Beijing, China) and P. pastoris GS115 (Invitrogen, Carlsbad, CA) were used for plasmid amplification and heterologous expression, respectively. The pEASY-T3 vector (TransGen) and pPIC9 (Invitrogen) were used for plasmid construction, respectively. Culture media for His+ transformants selection and P. pastoris growth and induction were prepared according to the manual of the Pichia Expression kit (Invitrogen). Mono-, di-, and trigalacturonic acid (GalpA, GalpA2 and GalpA3) and the substrate PGA were purchased from Sigma-Aldrich (St. Louis, MO). All other standard chemicals were of analytical grade.
5
Recombinant Cathepsin Proteolytic Assay
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Recombinant human cathepsins L, S and B were expressed in the methylotrophic yeast expression system P. pastoris (Invitrogen, Carlsbad, CA, USA) or in the E. coli expression system according to standard protocols [84 (link), 85 ]. Pure mature proteins were titrated with the broad spectrum cysteine cathepsin inhibitor E-64 yielding active concentrations of 120 µM, 48 µM and 96 µM for cathepsin B, L and S, respectively. The experiment was performed in phosphate buffer, containing 100 mM Na2HPO4, 1 mM EDTA and 1 mM DTT, pH 6.0. Recombinant cathepsins were added to the 14- 3- 3β protein (AbFrontier, Seul, Korea) at 1:100 enzyme/substrate molar ratio. Samples were then incubated at 37°C up to 120 min. As a negative control, E64 at a final concentration of 20 µM was added to block cathepsin activity. Samples were ran on 12.5 % SDS-PAGE electrophoresis gels and silver stained as described [86 (link)].
6
Purification and Analysis of Glycosidases
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Unless specified otherwise, ultrapure water (resistivity >18.0 megaohm-cm, Barnstead NanoPure Diamond) was used in the preparation of all buffers, substrates and enzyme assays. Pre-packed nickel nitrilotriacetic acid (Ni-NTA) was purchased from GE Healthcare (Little Chalfont, UK). All alcohols used in enzyme kinetics and product analysis were purchased from Sigma-Aldrich (St Louis, MO, USA) at 98% or higher purity. Peroxide contamination in commercial alcohols was determined using Quantofix, a semiquantitative dip-stick test from Sigma-Aldrich. Alcohols found to be contaminated with peroxide were passed through neutral alumina (previously activated by strong heating under flame) and re-tested. The glycosidases EndoH, EndoHf and PNGaseF were purchased from New England Biolabs (Ipswitch, MA, USA), while α-mannosidase was purchased from Sigma-Aldrich. Proteomics-grade trypsin was purchased from Promega (Madison, WI, USA). Galactose 6-oxidase from F. graminearum was expressed in P. pastoris (Invitrogen, Groningen, the Netherlands) and purified using pre-packed Ni-NTA29 (link). Bovine serum albumin and pre-cast 4–20% SDS–PAGE TGX were purchased from Bio-Rad (Hercules, CA, USA).
7
Upscaled Production of CorPerox
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The upscaled production of CorPerox was carried out in 1.3- and 7.5-liter bioreactors (New Brunswick BioFlo 115 fermentor, Eppendorf, Germany) as per the P. pastoris fermentation process guidelines (Invitrogen) with the following optimizations: The glycerol fed-batch phase was replaced by a sorbitol and methanol transition phase; besides, 200 μM (1.3-liter bioreactor) and 150 μM (7.5-liter bioreactor) of hemin were added to the methanol solution. CaCl2 (10 mM final) was added to the crude protein solution before being either directly purified or flash-frozen in liquid nitrogen and stored at −80°C. We verified that flash-freezing did not cause any activity loss, for both AlcOx and Perox enzymes.
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