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Thanks to everyone for the great suggestions so far. To clarify and answer a few questions, with the mutated construct we get no protein either secreted or in the pellet. The protein in question is about 20 Kda; with glycosylation at both sites it is around 29 kDa (both in mammalian cells and in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)