Human Fas ligand is a medically important transmembrane glycoprotein directing the induction of apoptosis. The influence of N-terminal part (Q103-P138) truncation of human Fas ligand extracellular domain (hFasLECD) on the expression of N-terminal FLAG-(Gly)(5)-tagged hFasLECD (NFG5-hFasLECD) with partial N-glycosylation-sites deletion in Pichia pastoris was investigated. The N-terminal part truncation significantly improved the secretion level of both singly (N184Q) and doubly (N184Q, N250Q) N-glycosylation-sites deleted NFG5-hFasLECD. The highly purified N-terminal truncated NFG5-hFasLECD with the double N-glycosylation-sites deletion mutation was obtained using single-step cation-exchange chromatography. The isolation yield was about 24mg from one liter culture supernatant, which amounted to approximately five times higher than that of the previously reported non-truncated NFG5-hFasLECD with N184Q mutation. The remaining N-linked carbohydrate chain in the purified product was digested with a high-mannose type glycochain specific endoglycosidase, Endo Hf, under non-denatured condition. The N-linked carbohydrate chain trimmed product was purified through Con A-agarose column fractionation and another cation-exchange chromatography from the reaction mixture. The final product showed the molecular weight exact to that of NFG5-hFasLECD-[Delta A(103-138), N250Q] mutant with single N-acetyl-glucosamine residue in MALDI-TOF mass-spectrometric analysis, and existed as a trimer in solution. The N-terminal truncated product either with or without N-linked carbohydrate chain exhibited the specific binding activity toward soluble human Fas receptor extracellular domain-human IgG(1)-Fc domain fusion protein, which revealed that the presence of N-linked carbohydrate chain was not essential for the functional activity of hFasLECD. The sample preparation system developed here may be applicable to the structural analysis of hFasLECD. (c) 2008 Elsevier Inc. All rights reserved.