Improved secretion of human Fas ligand extracellular domain by N-terminal part truncation in Pichia pastoris and preparation of the N-linked carbohydrate chain trimmed derivative

Abstract

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 24 mg 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-[Δ(103–138), N250Q] mutant with single N-acetylglucosamine 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.