title: Secretory expression of synthetic human Fas ligand extracellular domain gene in Pichia pastoris: influences of tag addition and N-glycosylation site deletion, and development of a purification method.
abstract: Human Fas ligand is a medically important membrane glycoprotein that induces the apoptosis of harmful cells. A new secretory expression and purification method was devised for the production of a large amount of recombinant human Fas ligand extracellular domain (hFasLECD) by Pichia pastoris. The expression plasmid containing a synthetic hFasLECD gene designed using yeast optimal codons was constructed for the secretion of hFasLECD. The secreted product exhibited the specific binding activity toward soluble human Fas receptor extracellular domain-human IgG(1)-Fc domain fusion protein, and the receptor-ligand complex was immunoprecipitated by Protein A conjugated agarose-gel beads. The influences of the N- and C- terminal addition of FLAG/(His)(6) tag spaced by pentaglycine sequence and the sequentially accumulative deletions of N-glycosylation sites within hFasLECD were investigated. The secretion of functional hFasLECD was retained after the N-terminal tagging and the deletion of either single or double N-glycosylation sites. As judged from SDS-PAGE analysis of the culture supernatant, the N-terminal addition of FLAG-(Gly)(5) tag and the deletion of single N-glycosylation site via N184Q mutation increased the secretion level of the product. In contrast, the C-terminal tagged genes and all N-glycosylation sites deleted gene failed to direct the secretion of functional hFasLECD. The secreted products in the culture medium were purified using a cation-exchange chromatography and a gel-filtration chromatography. The purified hFasLECDs existed as trimers composed of a mixture of monomer species in different glycosylation states. Approximately five milligram of functional N-terminal FLAG-(Gly)(5) tagged hFasLECD N184Q mutant was obtained from one liter culture supernatant.
authors.affiliation: Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan. m-muraki@aist.go.jp
authors.forename: Michiro
authors.initials: M
authors.lastname: Muraki
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D053222:Fas Ligand Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D053222:Fas Ligand Protein; D011993:Recombinant Fusion Proteins