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

BackgroundA 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.ResultsA 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.ConclusionsA 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.

Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene – methyltetrazine reactions

“…The production of NFK3G1CG4-hFasLECD in a P. pastoris secretory expression system was conducted as described previously [19]. hFasRECD-Fc was produced in a baculovirus – Bombyx mori expression system and purified as described in the previous paper [36].…”

Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation

“…1 ) as well as from the fairly hydrophilic property of the N-terminal FLAG tag plus aa 139-142 region (Asp–Tyr–Lys–Asp–Asp–Asp–Asp–Lys–Gly–Cys–Gly–Gly–Gly–Gly–Glu–Lys–Lys–Glu), the unpaired Cys was expected to locate not proximal to the binding interface, but exposed to the solvent in an aqueous solution of neutral pH. Although the pre-activation by a treatment with some reducing agents was necessary for an efficient conjugation because of the propensity to form a disulfide-bridge between two NFG1CG4-hFasLECD monomer subunits due to non-specific oxidation of the unpaired cysteine residue, it was demonstrated that selective activation of the conjugation site alone was possible by choosing the mild reducing condition using TCEP for the reaction in the previous study (Muraki 2014b ). Moreover, the resulting conjugates with several kinds of single or double maleimide group(s) containing compounds maintained the receptor binding activity, and also one conjugate was proved to exhibit significant cytotoxic activity against a cancer cell line after cross-linking by an anti-FLAG tag antibody (Muraki 2014b ).…”

“…hFasLECD containing single deletion mutation from 103 to 138 and double substitution mutations (N184Q and N250Q) [hFasLECD (139-281, N184Q, N250Q)] with an N-terminal FLAG-(GlyCysGlyGlyGlyGly) tag sequence (NFG1CG4-hFasLECD) and that with an N-terminal FLAG-(Gly) 5 tag sequence (NFG5-hFasLECD) were prepared as described (Muraki 2008 , 2014b ). FL-5Mal was obtained from Tokyo Chemical Ind.…”

“…The recombinant NFG1CG4-hFasLECD was produced by a Pichia pastoris secretion system. The sample used for the modification with FL-5Mal was pre-purified by cation-exchange chromatography (Hi-Trap SP HP) preceding the conjugation reaction as described (Muraki 2014b ), and protein concentration of the purified sample was determined to be 7.6 mg/ml by a BCA protein assay kit using bovine serum albumin as a standard. The solution (1.5 ml) containing 11.4 mg protein was first mixed with 30 μl of 0.5 M Ethylenediaminetetracetic acid sodium salt (EDTA Na) solution (pH 8.0) and then treated with 30 μl of 0.5 M TCEP solution (neutral pH), which gave a final reaction mixture containing 10 mM each of EDTA Na and TCEP.…”

“…On the other hand, conducting the chemical modifications raises the possibility of impairment of valuable biological activity of the original proteins, if randomly performed. In a recent study, the author developed a new method to introduce chemical modifications into an hFasLECD derivative via the conjugation of a reactive cysteine residue existing in the N-terminal tag sequence with a maleimide group containing compounds (Muraki 2014b ). The modifications were able to be carried out without damaging the original biological functions including apoptosis inducing activity of hFasLECD in the presence of a cross-linking antibody.…”