Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2004
Publication Date: 1/20/2004
Citation: Wu, S., Letchworth III, G.J. 2004. High efficiency transformation by electroporation of Pichia pastoris pretreated with LiAc and DTT. Biotechniques. 36:152-154.
Interpretive Summary: Yeast can be used to produce recombinant proteins. The yeast Pichia pastoris is particularly useful because it has an easily-controllable promoter. Vectors are available to insert recombinant genes into Pichia DNA for stabile expression, but few clones are produced by existing protocols. Numerous changes in the standard protocol resulted in a 200-fold increase in the number of clones produced. The improved protocol produces enough clones to allow the power of standard yeast genetic techniques, such as random mutation and selection for desirable characteristics, to be applied to this particularly useful yeast species.
Technical Abstract: Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast. We report here that pretreatment of Pichia pastoris with 0.1M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 200-fold. DTT alone enhanced the transformation efficiency up to 20-fold but LiAc alone had little effect. Cultures grown to 0.9 to 1.4 OD at 600 nm had higher transformation efficiencies than younger or older cultures. A cell concentration of 10(10) per milliliter gave the highest efficiencies. Digestion of pPIC9K within the AOX1 gene with Sac I gave efficiencies at least three times higher than digestion in other genes with other enzymes. Gentle washing of cells with 1M sorbitol following transformation enhanced the efficiency. Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kilovolts, 25 microFerrads, and 186 ohms. The transformation efficiency at optimal conditions reached 4.7x10(6)clones/ug DNA with pPIC9K. A maximum of 7.5x10(5) clones was produced when 1 ug of pPIC9K DNA was used.