Pgapzαb

A P. pastoris codon optimised nucleotide sequence (https://eu.idtdna.com/CodonOpt) encoding PA1 without the signal peptide (NCBI Accession P62930 residues 33–103), referred to as pea albumin full (PAF), and cloning primers were purchased from Integrated DNA Technologies (IDT). Restriction endonucleases were supplied by Thermo scientific or New England BioLabs. Electrophoresed DNA fragments were purified from excised gel slices using a Qiagen gel extraction kit. Plasmid DNA was prepared using Promega Wizard miniprep kits. T4 ligase kit was supplied by Promega. Phusion polymerase was from New England Biolabs. Pichia pastoris (SMD1168H strain), the expression vector pGAPZαB, and Easy comp Pichia transformation kit were from Invitrogen.
Anti-GNA antibodies were prepared by Genosys Biotechnologies, Cambridge, UK. Monoclonal 6x-His Tag Antibodies were from Fisher Scientific, UK. Secondary IgG horseradish peroxidase antibodies were from Biorad. Chemicals for chemiluminescence and buffer salts were supplied by Sigma.

A Pichia pastoris codon‐optimised nucleotide sequence encoding ω/κ‐hexatoxin‐Hv1h (accession no. S0F209; residues 38–76) hereafter referred to as HxTx‐Hv1h, and cloning primers were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA). Restriction endonucleases were supplied by Thermo Fisher Scientific (Waltham, MA, USA) or New England BioLabs (Ipswich, MA, USA). Electrophoresed DNA fragments were purified from excised gel slices using a Qiagen gel extraction kit. Plasmid DNA was prepared using Promega Wizard miniprep kits. T4 ligase kit was supplied by Promega. Phusion polymerase was from New England Biolabs. P. pastoris (SMD1168H strain), the expression vector pGAPZαB and Easy comp Pichia transformation kit were from Invitrogen (Thermo Fisher Scientific).
Anti‐GNA antibodies were prepared by Genosys Biotechnologies (Cambridge, UK). Monoclonal 6x‐His Tag Antibodies were from Thermo Fisher Scientific. Secondary IgG horseradish peroxidase antibodies were from BioRad (Hercules, CA, USA). Chemicals for chemiluminescence and buffer salts were supplied by Sigma Aldrich (St Louis, MO, USA).

Synthetic DNA constructs were synthesized by Epoch Biosciences (USA). The P. pastoris PIR1 gene (with its native secretion sequences)[16 (link)] was synthesized in-frame with the C. antarctica lipase B (LipB) and cloned into the constitutive expression vector pGAPZB (Invitrogen), containing the glyceraldehyde- 3 phosphate dehydrogenase (GAP) promoter to form pGAPZPIRLIPB. Likewise, the reporter egfp (enhance green fluorescent protein) gene was fused to the putative P. pastoris anchor FLO9 (Genome Net code F2QQL9-PICP7) and cloned into pGAPZαB (Invitrogen) to form pGAPZαFLO9GFP. Since it was unknown if FLO9 coded for a signal peptide it’s entire coding sequence was cloned in-frame with the S. cerevisiae α-factor secretion sequence present in pGAPZαB. Both plasmids were double-digested with NotI/XbaI and their released inserts were swapped. Transformants were plated in selective LB Low Salt medium containing 25 µg/mL zeocin. The resulting plasmids were named pGAPZPIRGFP and pGAPZαFLO9LIPB. All 4 plasmids (pGAPZPIRGFP, pGAPZαFLO9GFP, pGAPZPIRLIPB and pGAPZαFLO9LIPB) were linearized with AvrII (a site present with the GAP promoter) prior to transformation of P. pastoris X-33 (Invitrogen) cells via electroporation. Transformants were selected on YPD plates containing 100 μg/mL zeocin.