Pichia easycomp transformation kit

The native pro-protein gene for Tc-LysN was cloned in-frame with the S. cerevisiae’s α-mating factor secretory signal into pBSY2S1Z, under the control of the methanol-inducible AOX1 promoter, via golden gate cloning (Engler et al. 2008 ) using SapI restriction sites. Chemically competent E. coli DH5α cells were transformed with the resulting expression vector, pBSY2S1Z–Tc-LysN. Isolated recombinant plasmids from single-colony transformants, selected on low salt Luria-Bertani (LB) plates supplemented with 25 μg*mL⁻¹ Zeocin®, were sent for DNA sequencing to Genewiz (Leipzig, Germany).
Komagataella phaffii BG10 transformation was carried out by stringently following the guidelines of Invitrogen’s Pichia EasyComp™ Transformation Kit. In brief, 50 μL chemically competent Komagataella phaffii BG10 cells were transformed using ∼10 μg purified pBSY2S1Z–Tc-LysN that was linearized by SacI-HF (according to NEB’s protocol). Single colonies were screened for enzyme activity after ~24 h of induction in BMMY at 30 °C using the standard method outlined in Invitrogen’s Pichia EasyComp™ Transformation Kit. Tc-LysN activity was analyzed by employing the azocasein assay (“Determination of endopeptidase acting using the azocasein assay” section).

The pPICZαC_GA carrying the insert was linearized by SacI and transformed into P. pastoris GS115 (Invitrogen) competent cells. The competent cells were prepared by using the Pichia EasyComp Transformation kit according to the instruction guidelines (Invitrogen). Linearized vector pPICZαC, without insert, was also transformed into P. pastoris GS115 and used as a control. After transformation, Pichia cells were plated onto yeast extract peptone dextrose sorbitol (YPDS) agar plate containing 150 μg mL⁻¹ zeocin and incubated for 3 to 8 days at 30°C. Transformed colonies were confirmed by colony PCR using 5′AOX1 and 3′AOX1 primers and glucoamylase gene specific primers. Putative Mut⁺ colony was selected based on PCR using AXO1 primers. Several Mut⁺ colonies from YPDS agar plate were selected and independently blotted onto Buffered Methanol-complex Medium (BMMY) (containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB, 4 × 10⁻⁵% (w/v) biotin, 0.5% (v/v) methanol, and 100 mM potassium phosphate buffer pH 6.0) agar plate containing 1% (w/v) soluble starch. Glucoamylase producing transformants were identified by the formation of decolorization zone or clear halo around the Pichia colonies after the addition of iodine solution.

Human recombinant irisin (r‐irisin) was expressed and purified as previously described¹⁵. Briefly, human irisin cDNA (360 bp), designed and synthesized by Life Technologies, was cloned into the EcoRI/XbaI sites of the plasmid pPICZaA. According to the protocol provided with the PichiaEasyComp Transformation Kit (Invitrogen, Carlsbad, CA, USA), linearized pPICZaAirisin plasmid was used to transform Pichia pastoris X‐33, following which the yeast cells were cultured and r‐irisin expression was induced and r‐irisin protein in the culture supernatant was purified as described in a previous study (15).