Condensed Protocol for Competent Cell Preparation and Transformation of the Methylotrophic Yeast Pichia Pastoris

Abstract

The methylotrophic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous protein expression in part because of the simplicity of techniques required for its molecular genetic manipulation (1). Several different procedures are available for introducing DNA into P. pastoris-spheroplast generation (2), electroporation (3), alkali cation (3,4), or polyethylene glycol (PEG) treatment (5). Here we describe a condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection.The introduction of exogenous DNA into an organism requires two steps: (i) the preparation of competent cells for DNA uptake and (ii) the transformation of the cells with the DNA. Transformation of P. pastoris by electroporation is a quick procedure. However, preparation of conventional electroporation-competent cells requires hours of work involving several washes, incubations, and centrifugations. In contrast, competent cell preparation for the heatshock method is short, but transformation requires approximately 2 h (4). The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not work reliably.We have modified the preparation of competent cells from the heat-shock procedure (5) and combined it with transformation by electroporation (3) to yield a condensed protocol that works consistently with auxotrophic markers or antibiotic selection. The main modification of the heat-shock procedure is the addition of a step in which P. pastoris cells are incubated in an optimized concentration of dithiothreitol (DTT). The cells prepared by this "hybrid" method are then electroporated using the same parameters as conventional electroporation.Transformation efficiencies using the condensed protocol are comparable to the conventional electroporation procedure using auxotrophic markers but are approximately 20-fold lower using the zeocin resistance marker. However, the condensed protocol provides sufficient transformants, including multicopy integrants, for protein expression studies and has several advantages over the conventional electroporation and heat-shock methods. Table 1 compares the steps in cell preparation and transformation for conventional electroporation, heat shock, and our condensed protocol. Compared to the heat-shock method, the condensed protocol requires less time for the transformation step and provides much higher transformation efficiencies. Compared to the electroporation procedure, the new procedure saves both reagents

Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 α-Glucosidases

“…1) is often found in subfamilies 1,2,5,15,17,19,24,27,28,31,32, and 36, which include -1,4-linkage-specific enzymes such as -amylases, cyclomaltodextrin glucanotransferases, and -glucosidases. Most -amylases and cyclomaltodextrin glucanotransferases have dipeptide Lys-His at the C-terminal end of the motif.…”

“…The expression plasmids were linearized by restriction digestion with SacI and used in the transformation of P. pastoris GS115. Competent cells were prepared by the method of Lin-Cereghino et al, 19) with some modifications. Briefly, P. pastoris was grown in 10 mL of YPD (1% yeast extract, 2% peptone, and 2% glucose) until A 600 reached 1.…”

“…3,13) For instance, -amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41), cyclodextrin glucanotransferase (EC 2.4.1. 19), and neopullulanase (EC 3.2.1.135) are GH-13 enzymes. Exo-glucoside-acting enzymes, such as oligo-1,6-glucosidase (EC 3.2.1.10), dextran glucosidase (EC 3.2.1.70), amylosucrase (EC 2.4.2.4), sucrose phosphorylase (EC 2.4.1.7), and isomaltulose synthase (EC 5.4.99.11), are also included in the family.…”

Multiple RIBEYE–RIBEYE Interactions Create a Dynamic Scaffold for the Formation of Synaptic Ribbons

“…Pichia pastoris yeast strain GS115 (his4 ) (Invitrogen) was used for heterologous protein expression (Lin-Cereghino et al, 2005). Yeast cultures were grown at 30°C on synthetic minimal medium containing 0.67% yeast nitrogen base (without amino acids supplemented with ammonium sulfate and appropriate amino acid-base; Formedium).…”

Expression and One-Step Purification of Recombinant Proteins Using an Alternative Episomal Vector for the Expression of N-Tagged Heterologous Proteins inPichia pastoris

“…7) After transformation, the cells were grown in YPDS þ Z plates (1% yeast extract, 2% peptone, 2% glucose, and 1 M sorbitol) containing 1.5% agar and zeocin (100 mg/mL) for 3 or 4 d at 30…”