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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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Muraki M

doi

10.1016/j.pep.2006.08.006

subject

Has Abstract

pub_date

2006-12-01 00:00:00

pages

137-46

issue

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(06)00254-3

journal_volume

pub_type

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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.