Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants.

Pichia pastoris is a yeast capable of expressing large amounts of some proteins. When expression vectors are introduced into P. pastoris, individual transformants typically express widely varying amounts of protein. Because clones expressing the highest level of protein occur infrequently during the transformation process, finding them can be very labor-intensive. We developed an immunological based filter screening method that rapidly detects transformants secreting large amounts of a heterologous protein. We have applied this method to the expression of a soluble trimeric form of CD40L, a molecule that regulates B-cell responses. Using this method, we identified transformants with one to 13 copies of the CD40L expression cassette. Maximum expression was obtained with clones containing eight or more copies of the expression cassette, and a clone with eight copies was selected for further analysis. High cell density fermentation of this clone using a mixed glycerol:methanol feed yielded 255 mg CD40L per liter of supernatant.