title: Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications.
abstract: BACKGROUND
  A useful heterologous production system is required to obtain sufficient amounts of recombinant therapeutic proteins, which are often necessary for chemical characterization and engineering studies on the development of molecules with improved properties. Human Fas ligand extracellular domain (hFasLECD) is an agonistic death ligand protein that has potential applications for medical purposes. Site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. This study aimed to enhance the yield of hFasLECD using a Pichia pastoris secretory expression system suitable for efficient production on a small laboratory scale, and further to provide procedures for its site-specific chemical modification without impairing the biological functions based on the developed production system.
  
  
  RESULTS
  A convenient cultivation system using a disposable plastic bag provided a three-fold increase in purification yield of tag-free hFasLECD as compared with the conventional system using a baffled glass flask. The system was further applied to the production of a mutant, which contains an additional reactive cysteine residue in the N-terminal tag-sequence region. Site-specific conjugations and cross-linking without impairing biological functions were achieved by reaction of the mutant hFasLECD with single maleimide group containing compounds and a linear polyethylene glycol derivative containing two maleimide groups at either end, respectively. All purified tag-free and chemically modified hFasLECDs showed an evident receptor binding activity in co-immunoprecipitation experiments mediated by wild-type and N-glycosylation site deficient mutant human Fas receptor extracellular domain derivatives. An N-Ethylmaleimide conjugated hFasLECD derivative demonstrated a significant cytotoxic activity against human HT-29 colorectal cancer cells.
  
  
  CONCLUSIONS
  A new, efficient cultivation system for enhanced secretory production of hFasLECD using P. pastoris and an effective strategy for site-specific chemical modifications of hFasLECD were devised. The results obtained constitute the basis for biomedical applications including developments of novel therapeutic proteins and diagnostic tools targeted to related diseases and their biomarkers.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan. m-muraki@aist.go.jp.
authors.forename: Michiro
authors.initials: M
authors.lastname: Muraki
mesh_terms: D053222:Fas Ligand Protein / Q000096:biosynthesis*; D019073:HT29 Cells; D006801:Humans; D010843:Pichia / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D053222:Fas Ligand Protein; D011994:Recombinant Proteins