Pichia transformation

A method for transforming whole cells of Pichia pastoris with a desired DNA, which method comprises:

• (1) providing a first suspension of from 5.times.10.sup.7 to 5.times.10.sup.8 competent cells/ml in an aqueous solution having a concentration between about 50 mM and about 500 mM of a non-toxic alkali metal salt selected from LiCl and Li.sub.2 SO.sub.4 and buffered to a pH between about 6 and about 8;
• (2) making a second suspension of competent cells by combining (a) said first suspension of said competent cells in an aqueous solution comprising the same alkali metal salt as used in step (1) and (b) a third aqueous solution with a volume less than 0.2.times. that of said suspension, said third aqueous solution being buffered at a pH between about 7 and about 8 and comprising between about 1 .mu.g and about 20 .mu.g of desired DNA per ml of said first suspension combined with said third solution;
• (3) incubating at least a portion of said second suspension made in step (2) at a temperature between about 27.degree. C. and about 33.degree. C. for a period between about 10 minutes to 60 minutes;
• (4) making a third suspension of said cells by mixing, to establish substantially homogenous polyethylene glycol concentration in said third suspension, at least a portion of the portion of said second suspension, incubated according to step (3), with a fourth aqueous solution, which has a volume between about 5 and about 10 times the volume of the portion of said incubated second suspension with which the fourth solution is mixed, said fourth solution consisting essentially of between about 20% (w/v) and about 50% (w/v) polyethylene glycol of average molecular weight between about 3000 and about 5000 daltons in a solution with the same composition as said second solution;
• (5) incubating at least a portion of said third suspension made in step (4) at between about 27.degree. C. and about 33.degree. C. for at least about 10 minutes to about 60 minutes;
• and
• (6) heat shocking at least a portion of the portion of said third suspension incubated according to step (5).