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
© Muraki; licensee BioMed Central Ltd. 2014
Received: 25 June 2013
Accepted: 3 March 2014
Published: 11 March 2014
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.
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.
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.
KeywordsHuman Fas ligand Extracellular domain Production Pichia pastoris Yield enhancement Site-specific chemical modification
Abnormal apoptosis can cause many serious diseases, such as cancers and rheumatoid arthritis, in the human body . Therefore, it has been assumed that the successful artificial control of apoptotic processes will play a substantial role in therapeutic interventions of these diseases [2, 3]. Human Fas ligand is a major death ligand protein, which triggers the execution of cellular apoptosis via extrinsic pathway by specific binding of its extracellular domain to that of an agonistic molecule, human Fas receptor . Heterologous expression is an important means to fulfill the requirements for obtaining sufficient amounts of therapeutic human proteins. Developing an efficient production system is often necessary not only for direct utilization as practical medicines, but also for chemical characterization and engineering studies [5, 6]. Accordingly, functional recombinant wild-type human Fas ligand extracellular domain (hFasLECD) and its derivatives have been produced in heterologous systems using several kinds of expression hosts including Escherichia coli , Pichia pastoris [8–10] and Dictyostelium discoideum .
Chemical modification, represented by pegylation, is a powerful method for the development of engineered proteins with beneficial functions, which include prolonged therapeutic activity in circulating blood, by adding specific chemical properties into the target protein molecules . It may be also useful for the structural characterization of interesting biological functions in native proteins. However, non-specific chemical modifications of protein molecules can interfere with the expression of their intrinsic therapeutic properties either by direct chemical transformation or by physical coverage of the critical functional groups. Site-specificity in the modification can contribute to the enhancement of useful functions and avoid unwanted effects of chemical modifications. In this connection, we have conducted several site-specific chemical modification studies with human lysozyme for the purpose of both the alteration of substrate specificity  and the clarification of the origin of carbohydrate recognition specificity .
The extracellular domain of human Fas ligand locates at the carboxyl-terminal region [amino acid residues (aa) 103-281] of whole molecule consisting of 281 aa, and independently exists as trimeric subunits without the help of other parts of the molecule under physiological conditions . This domain contains three N-glycosylation sites (Asn 184, Asn 250 and Asn 260) and two cysteine residues (Cys 202 and Cys 233) forming a disulfide-bridge. In previous studies, we developed a secretory production system of recombinant hFasLECD using P. pastoris as the expression host, and reported that both the addition of N-terminal FLAG®-(Gly)5 tag sequence  and the deletion of the non-essential region in trimerization (aa 103-138)  significantly increased the secretion level of the products. We also showed that two asparagine residues (Asn 184 and Asn 250) could be mutated to glutamine residues without serious reduction of the secretion level , and that the remaining heterogeneous N-glycan chains attached to Asn 260 in the N-terminal FLAG®-(Gly)5 tagged double N-glycosylation sites mutant could be trimmed to homogeneous N-acetyl glucosamine residues without impairing the binding activity  toward a recombinant human Fas receptor extracellular domain (hFasRECD) derivative produced in silkworm larvae [14, 15].
In this report, a marked increase in the production yield of tag-free hFasLECD achieved by the utilization of a disposable plastic bag as the cultivation vessel is described. This system was further applied to the secretory production of a mutant, which has an additional reactive cysteine residue within the above mentioned N-terminal FLAG®-(Gly)5 tag sequence. The details of site-specific chemical modifications of this mutant with maleimide group containing compounds as well as the characterizations of the purified reaction products concerning binding activity toward hFasRECD and cytotoxic activity against a cancer cell line will be presented.
Enhanced yield of tag-free hFasLECD using disposable culture-bag
Purification course of tag-free hFasLECD and its N-glycan trimmed derivative
Sample volume (ml)
Total protein (mg)
1st Ultrafiltration plus buffer exchange
1st Cation-exchange chromatography and 2nd ultrafiltration plus buffer exchange
2nd Cation-exchange chromatography
Endo Hf treatment, Con A-column fractionation and 3rd Cation-exchange chromatography
As demonstrated in the previous study concerning FLAG®-(Gly)5 tagged sample , the remaining N-glycans in tag-free hFasLECD sample in this study could also be trimmed with an Endo H-type glycosidase, Endo Hf (Figure 2d, lane b). In Figure 2b and Figure 2c, the 3rd cation-exchange chromatography profile of the sample after the digestion with Endo Hf and the size-exclusion chromatography profile of the cation-exchange chromatography fractionated sample are shown, respectively. The sample-peak elution-time in the size-exclusion chromatography was substantially delayed after Endo Hf digestion (Figure 2c), which showed the effect of N-glycan trimming on the molecular weight of tag-free hFasLECD. Figure 2d summarizes the purification course during the N-glycan trimming using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (lanes a-d).
Secretory expression of NFG1CG4-tagged hFasLECD
Chemical modification of NFG1CG4-hFasLECD with single maleimide group containing compounds
In order to probe the assembly state and the molecular shape of each modified product, the fractionated samples in the above cation-exchange chromatography were subjected to analysis using a size-exclusion chromatography (Figure 7b). Either sample presented a symmetrical single peak, suggesting the uniformity of molecular-weight of the fractionated products (Figure 7b, center and right panels). The peak retention time of the N-Ethylmaleimide conjugated sample (28.78 min) was between the size-markers of 44 kDa (30.22 min) and 158 kDa (25.04 min) (Figure 7b, left panel), and was nearly the same as observed for tag-free hFasLECD (29.55 min) under the identical elution condition using the same chromatography column (Figure 2c, solid line). This indicated that no significant change in the molecular conformation occurred to hFasLECD by the conjugation of N-Ethylmaleimide. On the other hand, SUNBRIGHT® ME-050MA conjugated sample eluted markedly early (22.26 min) and the peak retention time was even significantly earlier than the 158 kDa size-marker (25.04 min). This suggested that the PEG moieties attached to the N-terminal tag sequence region of NFG1CG4-hFasLECD had a much more extended conformation to behave as a molecule with virtually bigger molecular weight, which rendered the retention time markedly earlier than the expected value from its actual calculated molecular-weight (ca. 70 kDa as the triply modified product).
Purification course of NFG1CG4-hFasLECD conjugated with single maleimide group containing compounds
Sample volume (ml)
Total protein (mg)
1st Ultrafiltration plus buffer-exchange
1st Cation-exchange chromatography and 2nd ultrafiltration plus buffer-exchange
After chemical modification reaction and 2nd cation-exchange chromatography*2
The purified samples conjugated with single maleimide group containing compounds exhibited comparable binding activity to that of NFG5-hFasLECD toward wild type hFasRECD-T-Fc (Figure 4a, lanes a, c and d). Both bands in the SUNBRIGHT® ME-050MA conjugated sample were found in the materials co-immunoprecipitated with wild-type hFasRECD-T-Fc protein. Therefore, the minor band was considered to be derived from a small fraction of the functional trimeric product containing the subunit component with the remaining non-conjugated cysteine residue. The comparison of the data using wild-type hFasRECD-T-Fc with that of the N-glycan deficient hFasRECD-T-Fc mutant revealed that the binding activity toward this mutant was essentially maintained with all examined hFasLECD samples (Figure 4b), however the SUNBRIGHT® ME-050MA conjugated sample had a reduced binding strength toward the mutant hFasRECD-T-Fc than the wild-type protein judging from the evident but weaker density of the bands of the co-immunoprecipitated materials (Figure 4b, lanes e and f).
Cross-linking of NFG1CG4-hFasLECD with two maleimide-groups containing polyethylene glycol
Cytotoxic activity of chemically modified NFG1CG4-hFasLECD
Heterologous production systems of recombinant human Fas ligand extracellular domain have been developed using several expression hosts. It has been shown that P. pastoris and D. discoideum were the most efficient hosts in providing functional products by direct secretion without the necessity of fusion with other protein domains . A methanol utilization plus strain, P. pastoris GS115 (Mut+), has been used as the host in the secretory production of this protein [8–10, 14]. The best purification yield of 24.3 mg/l was reported with the NFG5-hFasLECD (aa 139-281) containing the mutations of N184Q and N250Q . However, the deletion of NFG5 tag sequence in this mutant resulted in a large reduction of the purification yield to 4.2 mg/l . In this study, it was shown that the purification yield of tag-free hFasLECD could be increased 3-fold by altering the cultivation vessel used in the production system from glass baffled flask to disposable plastic bag. Good aeration during cultivation in expression induction-phase using methanol is a critical factor in the production efficiency of the recombinant proteins in P. pastoris. The cell density of another species of yeast, Saccharomyces cerevisiae, was significantly increased in cultivation using the plastic culture-bag compared with that using baffled flask (Y. Kawabata, personal communications). In another experiment for the production of NFG1CG4-hFasLECD in P. pastoris GS115 strain using 4000 ml BMMY medium in a 10 liter volume plastic culture-bag (CB-10; Fujimori Kogyo), an approximately 50% increase in optical density at 600 nm was obtained compared with the corresponding cultivation using 50 ml BMMY medium in 500 ml triple side-baffled glass flask after 96 h cultivation (unpublished result). Therefore, the main reason for the yield enhancement in this study may be ascribed to better aeration by the forced air ventilation using a diaphragm pump and increase of cell density in the cultivation.
Single-use technologies using disposable apparatus including plastic bags are currently evolving industrial technologies, which have a number of merits in the production of recombinant proteins, especially in the field of bio-pharmaceutical manufacturing . One of the important areas of the application of single-use technologies is fermentation of microorganisms including P. pastoris. In this study, it was demonstrated that the use of a disposable plastic bag was also effective for the purpose of enhancing the yield of recombinant proteins aiming at the research of its site-specific modification using P. pastoris as the producer. To date, remarkable increases in the yield of recombinant proteins from shake-flask systems have been achieved in many cases by non-disposable system such as a jar fermenter, which are mostly made of glass and stainless-steel . This study demonstrated that the alternative possibility of enhancing the yield using disposable plastic culture-bag system, though the increased level was moderate as compared with a reusable fermenter system operated under optimized conditions. Alteration of the cultivation vessel provided not only an increase in the purification yield per unit volume of the culture supernatant but also reduction of the number of vessels necessary for the same total cultivation volume. Moreover, the plastic culture-bag system requires neither any special ingredient in the medium nor expensive equipment for cultivation, making this system convenient compared with a fermenter system, which requires special materials for cultivation. The culture-bag system should be readily applicable to production of other recombinant proteins in yeasts on a small laboratory scale.
The interactions between hFasLECD and hFasRECD constitute a key signaling step in the initiation of apoptotic processes via extrinsic pathway mediated by human Fas receptor. Some aspects of the interactions have been characterized through the site-directed mutagenesis studies [19, 20] and the three-dimensional structures produced by in silico modeling [21, 22]. Either tag-free hFasLECD containing untrimmed or trimmed N-glycan exhibited virtually identical binding activity toward mutant hFasRECD-T-Fc lacking N-glycosylation site within the hFasRECD part with the activity toward wild type hFasRECD-T-Fc, suggesting N-glycosylation in both hFasLECD and hFasRECD is not a prerequisite for protein – protein interactions between hFasLECD and hFasRECD.
Site-specific chemical modifications provide a useful method to obtain proteins with improved pharmaceutical properties by enhancing in vivo biological efficacy. A number of therapeutic proteins with site-specific modifications, especially pegylation, have been developed . Suitable selection of the structural position for modification is essential for effective derivation, while maintaining the intrinsic biological functions of the target proteins. The N-terminal region of NFG1CG4-hFasLECD is thought to be located distant from the binding interface to hFasRECD judging from a detailed three-dimensional structure of the complex between hFasLECD and a decoy receptor, DcR3 .
NFG5-hFasLECD (aa 139-281, N184Q, N250Q) is considered to contain a single disulfide bond between Cys 202 and Cys 233 within a structurally buried region by analogy with the available three-dimensional structure of hFasLECD . However, it is known that TCEP can selectively reduce solvent exposed disulfide bonds under mild reaction conditions without affecting the structurally buried one . Therefore, the N-terminal tag sequence region in NFG5-hFasLECD  was chosen as the introduction site of solvent accessible cysteine residue for site-specific chemical modifications in this study. A free cysteine residue is ideal for selective modifications due to its unique reactive properties against many electorophiles [25, 26]. The maleimide group containing compound is useful, since a variety of commercial compounds including those for pegylation are readily available with reasonable prices. It was found that NFG1CG4-tag sequence, FLAG®-GlyCys(Gly)4, effectively worked as a new N-terminal tag for site-specific chemical modifications, which provided an alternative to existing methods for N-terminal conjugation using different tag sequences [27, 28].
The N-Ethylmaleimide conjugated NFG1CG4-hFasLECD exhibited a cell-death inducing activity against HT-29 cells, a human colorectal adenocarcinoma cell line. The cytotoxic activity was significant only after cross-linking by M2 antibody specific for FLAG®-tag as depicted with the activity of a FLAG®-tagged trimeric hFasLECD against Jurkat, Raji and HeLa cells . This suggested that site-specific chemical modifications via conjugation of the cysteine residue in the N-terminal NFG1CG4-tag with maleimide compounds were possible without losing the intrinsic pro-apoptotic function of the sample produced in P. pastoris . This result constitutes the basis of development of novel type cytotoxic therapeutic agents using site-specific chemical modifications with targeting moieties such as tumor antigen specific single chain antibody [30, 31]. This also showed that the two N-glycan chains at Asn184 and Asn250 sites were not always essential for exhibiting the cell-death inducing activity.
To our knowledge, this is the first report describing preparations of functional hFasLECD samples with site-specific chemical modifications. Human FasLECD works as a pro-apoptotic receptor agonist by binding to hFasRECD on the plasma membrane of target cells. However, it also binds to DcR3 with a similar affinity to hFasRECD at the same time , which can be one of the possible reasons leading to onset of diseases caused by excessive cellular proliferation . Overexpression of DcR3 decoy receptor was found in several types of cancers and autoimmune diseases . Protein engineering studies including the site-specific chemical modifications might contribute to solve this problem by tuning the receptor binding specificity of hFasLECD.
It has been shown that a hexameric, genetically fused proteins containing two trimers of hFasLECD within the assembled molecule via the association of ACRP30 collagen domain region, displayed much higher killing activity than trimeric hFasLECD against several kinds of tumor cells . The site-specific cross-linking strategy concerning hFasLECD demonstrated in this study might also contribute to the development of such engineered molecules with enhanced cell-killing activity.
Possible applications aimed at membrane bound forms of human Fas receptor on the targeted cells include in vivo / in vitro imaging of positive cells  by conjugations with either fluorescent dyes  or luminescent proteins . It is also known that soluble agonistic and decoy receptor proteins concerning human Fas receptor system are useful biomarkers in serum, urine and other body fluids for early diagnosis , prognosis [39, 40], response to drug treatment  and mortality  of many serious human diseases represented by cancers. The mutant hFasLECD containing the reactive cysteine residue conjugable to maleimide group containing compounds should also become a powerful molecular agent in developing devices for quantifying such disease specific biomarkers.
In the present study, a new, convenient and efficient production system by P. pastoris using a disposable plastic culture-bag was developed, which requires neither special ingredients in the culture medium nor expensive equipment such as a jar fermenter. Using this system, the purification yield of tag-free hFasLECD increased three-fold. This system was also applicable to the secretory production of a mutant hFasLECD, which was appropriate for the site-specific conjugations with maleimide group containing compounds. The enhanced yield will facilitate further chemical characterization studies on hFasLECD. The conjugated hFasLECDs with maleimide group containing compounds at its N-terminal tag sequence showed receptor binding and cell-death inducing activities. The site-specific chemical modifications of hFasLECD should contribute to development of novel therapeutic agents as well as tools for diagnostic purposes in the biomedical field.
Plasmid vectors, pNFG5-hFasLECD (aa 139-281) with (N184Q, N250Q) double mutations and its tag-free version were prepared as described in previous papers [10, 14]. The insertion mutation for the introduction of the additional cysteine residue in NFG1CG4-hFasLECD (Figure 1a) was conducted by in vitro mutagenesis of NFG5-hFasLECD gene as a custom service by Takara-bio Co. Pichia pastoris GS115 (Mut+) was used as the strain for expression. Buffered glycerol complex medium (BMGY medium) and buffered methanol complex medium (BMMY medium) were prepared as described . Triple side-baffled culture flasks were purchased from Asahi Glass Co., Ltd. Disposable plastic culture-bags (CB-5) and a stainless-steel bag-holder (Bag-holder 10) were products of Fujimori Kogyo Co., Ltd. NFG5-hFasLECD protein was obtained as described previously . Wild-type and (N102Q, N120Q) mutant hFasRECD-T-Fc proteins were obtained using baculovirus-silkworm expression system . The 10-20% gradient gels used for SDS-PAGE analysis and L-Cysteine hydrochloride monohydrate were purchased from Wako Pure Chemical Ind., Ltd. Culture filtration-devices and tangential flow filtration-devices for concentration were obtained from Nihon Pall, Ltd. Cation-exchange chromatography and size-exclusion chromatography were performed using columns and devices from GE healthcare. BCA protein assay kit and TCEP neutral pH solution were purchased from Thermo Fisher Scientific Inc. SUNBRIGHT® ME-050MA: α-[3-(3-Maleimido-1-oxopropyl) amino]propyl-ω-methoxy, polyoxyethylene (5 kDa fraction, 98.1%; average molecular weight, 5393; polydispersity, 1.02; maleimide group content, 95.0%) and SUNBRIGHT® DE-100MA: α-[3-(3-Maleimido-1-oxopropyl)amino]propyl-ω-[3-(3-Maleimido-1-oxopropyl)amino]propoxy, polyoxyethylene (10 kDa fraction, 96.0%; average molecular weight, 10644; polydispersity, 1.02; terminal activated rate, 83.9%) were obtained from NOF Co. N-Ethylmaleimide and Phosphate buffer solution (pH 6.4) were from Nakarai Tesque. Immunoprecipitation kit (Protein A) was from Roche Diagnostics. Other chemical reagents of analytical grade and devices used for protein purification were as described . Chemical structure of maleimide group was drawn using Accelrys Draw 4.1.
Production of tag-free hFasLECD using baffled glass culture-flask
The experimental procedures used during the selection of efficient single colonies of the recombinant P. pastoris and those used for the secretory production of tag-free hFasLECD in 500 ml scale culture of BMMY medium (pH 6.2-6.5) using a 3000 ml baffled flask made of borosilicate glass were the same as described previously . Cultivation was conducted at 29.5°C with a rotation of 300 rpm in a thermostatic air incubator (BR-32FL; TAITEC).
Expression of tag-free and NFG1CG4-hFasLECDs using disposable plastic culture-bag
The same BMMY medium for the above baffled culture-flask was used in the cultivation of P. pastoris transformant using a disposable culture-bag made of polypropylene. The P. pastoris pre-culture was prepared by cultivation in 500 ml BMGY medium (pH 6.2-6.5) at 29.5°C, 300 rpm overnight using 3000 ml triple side-baffled glass flask. The pre-culture was centrifuged at 8000 rpm for 2 min at room temperature to give the seed cell-pellets for the inoculation of 2500 ml BMMY medium made of either autoclaved or filter-sterilized components in 5000 ml volume of the disposable culture bag. The cultivation was conducted at 29.5°C with a rotation of 80-85 rpm for 96 h in the same thermostatic air incubator as described above. The induction of expression was made by the addition of 0.5% methanol at 24 h intervals.
Purification of secreted products
The culture medium containing the secreted hFasLECDs was centrifuged and the supernatant was sterilely filtered with a double (0.8 μm and 0.2 μm) poly-ethersulfone membrane. The filtrate was transferred to an ultrafiltration device equipped with a poly-ethersulfone membrane for tangential flow filtration (Molecular-weight cut off: 10 kDa) to concentrate to approximately 100 ml. The concentrated retentate was further buffer-exchanged using 50 mM sodium acetate (pH 5.6). The buffer-exchanged solution was then loaded on a Hi-Trap S cation-exchange column (5 ml) equilibrated with 50 mM sodium acetate buffer (pH 5.6). The recombinant protein was eluted with either 500 mM NaCl (for tag-free hFasLECD) or 300 mM NaCl (for NFG1CG4-hFasLECD). The eluted samples were concentrated using an Amicon Ultra-15 ultrafiltration device (10 kDa) to ca. 4 ml, and further desalted with PD-10 column (8.3 ml) using 50 mM sodium acetate buffer (pH 5.6).
With respect to tag-free hFasLECD, the desalted solution was loaded on a Resource S cation-exchange column (6 ml) equilibrated with 50 mM sodium acetate buffer (pH 5.6). The recombinant proteins were eluted with a linear salt gradient from 50 to 450 mM NaCl in 50 mM sodium acetate buffer (pH 5.2) at the flow rate of 6 ml/min. The fractions containing the recombinant hFasLECD were pooled as the final product. The protein concentration of the samples at each purification step was determined by a BCA protein assay kit using bovine serum albumin as a standard. As for NFG1CG4-hFasLECD, the protein concentration of the desalted solution after the purification step with the Hi-Trap S 5 ml column was determined to be 9.9 mg/ml, and was directly used for the reaction with maleimide group containing compounds.
Preparation of N-glycan trimmed tag-free hFasLECD
Purified sample of tag-free hFasLECD was concentrated to 1.92 mg/ml with Amicon Ultra 15 (Molecular weight cut-off: 10 kDa), and digested with Endo Hf (New England Biolabs, Inc.). Twelve μl of 500 mM sodium citrate buffer (pH 5.5) was added to 192 μg of tag-free hFasLECD sample and then treated with 12000 U of Endo Hf at 37°C, for 48 h. The N-glycan trimmed tag-free hFasLECD in the reaction mixture was purified according to essentially the same procedures as described for NFG5-hFasLECD . In brief, the above Endo Hf reaction mixture was first subjected to Con A-agarose column to trap the N-glycan untrimmed tag-free hFasLECD, and the flow-through fraction containing the N-glycan trimmed tag-free hFasLECD was then further fractionated by Mono S 1 ml column cation-exchange chromatography using the elution with a linear salt gradient from 50 to 550 mM NaCl in 50 mM sodium acetate buffer (pH 5.6).
Reaction of NFG1CG4-hFasLECD with maleimide group containing compounds
The reactions of NFG1CG4-hFasLECD sample with single maleimide group containing compounds (N-Ethylmaleimide and SUNBRIGHT® ME-050MA) were conducted as follows. An aliquot of the sample solution (1.6 ml) containing 15.8 mg protein was mixed with 16 μl of 0.5 M Ethylenediaminetetraacetic acid sodium salt (EDTA Na) solution (pH 8.0) and then treated with 64 μl of 0.5 M TCEP solution (neutral pH) for 1 h at 27°C. The reaction mixture was resolved by a PD-10 desalting column (8.3 ml) to remove excess amount of TCEP using 25 mM phosphate buffer plus 2 mM EDTA Na (pH 6.4) as the eluent. After the loading of the sample in a total volume of 2.5 ml into the column, 3.5 ml of the buffer followed by another 2.5 ml of the buffer was added to collect the flow-through. The former 3.5 ml and the latter 2.5 ml flow-through fractions were named “Elution fraction” and “Wash fraction”, respectively. Virtually, no reduced product was found in the “Wash fraction” (Figure 6b, lane d). The “Elution fraction” was divided into two 1.75 ml parts equally. Either freshly prepared 30 μl of 1 M N-Ethylmaleimide solution in ethanol or 189 mg of solid SUNBRIGHT® ME-050MA powder was added to each solution. After 15 min at 27°C, 33 μl of 1 M L-Cysteine hydrochloride solution in water was added to each reaction mixture, and further incubated at 27°C for 15 min in order to quench the excess maleimide groups.
The reaction of NFG1CG4-hFasLECD sample with SUNBRIGHT® DE-100MA was performed as follows. An aliquot of the sample solution (1.6 ml) containing 15.8 mg protein was first treated with TCEP in the same way as described for the reaction with single maleimide group containing compounds. Then, 100 μl of freshly prepared SUNBRIGHT® DE-100MA solution (80 mg in 5 ml) in 25 mM phosphate buffer plus 2 mM EDTA Na (pH 6.4) was added to the PD-10 column “Elution fraction” (3.5 ml), and incubated for 1 h at 22°C. Seventy μl of 1 M N-Ethylmaleimide solution in ethanol was added to the reaction mixture and was incubated for 15 min at 22°C to cap the unreacted free cysteine residues in the NFG1CG4-hFasLECD sample. After that, 69 μl of 1 M L-Cysteine hydrochloride was added and incubated for a further 15 min at 22°C to quench the excess maleimide groups.
Purification of chemically modified NFG1CG4-hFasLECD
The final reaction mixtures concerning the chemical modification of NFG1CG4-hFasLECD with single maleimide group containing compounds were buffer-exchanged with 50 mM sodium acetate solution (pH 5.3) using PD-10 desalting column (8.3 ml). This solution was loaded on a Resource S cation-exchange column (1 ml) equilibrated with the same buffer. The recombinant protein was resolved with a linear salt gradient from 0 to 250 mM NaCl in 50 mM sodium acetate buffer (pH 5.3) at the flow rate of 1 ml/min. The main peak fractions containing the chemically modified NFG1CG4-hFasLECD were pooled, and the product yield was quantified by BCA protein assay.
As for the reaction product of NFG1CG4-hFasLECD with SUNBRIGHT® DE-100MA, the final reaction mixture was first desalted using PD-10 column (8.3 ml) and then concentrated to ca. 1.0 ml with Amicon Ultra 8 ultrafiltration device (Molecular-weight cut off: 10 kDa). An aliquot (230 μl) of the concentrated sample was resolved by a Superdex 200 10/30 GL size-exclusion column (diameter, 10 mm; length, 300 mm) using 50 mM sodium acetate plus 150 mM NaCl (pH 5.6) elution buffer at the flow rate of 0.5 ml/min. The samples of each peak fraction were analysed by SDS-PAGE. The second and the third peak fractions containing inter-molecularly cross-linked NFG1CG4-hFasLECD products were pooled, and the recovery yields were quantified by BCA protein assay.
Molecular-weight estimation using size-exclusion chromatography
Molecular-weight of the purified samples was estimated by a Superdex 200 10/30 GL size-exclusion column (diameter, 10 mm; length, 300 mm) using the same elution conditions as described above. Fifty five μg each of the untrimmed and N-glycan trimmed tag-free hFasLECD, 35 μg each of the NFG1CG4-hFasLECD modified with single maleimide group containing compounds (N-Ethylmaleimide and SUNBRIGHT® ME-050MA), or 230 μl each pooled fractions of the NFG1CG4-hFasLECD cross-linked with SUNBRIGHT® DE-100MA were subjected to chromatography. A mixture of molecular-weight standard proteins (Aldolase, 158 kDa; Ovalbumin, 44 kDa and Ribonuclease A, 13.7 kDa) was used as size-markers.
Receptor-mediated co-immunoprecipitation of hFasLECD
Detection of the binding activity of hFasLECD samples toward Fas receptor was performed by receptor-mediated ligand immunoprecipitation assay using hFasRECD-T-Fc proteins and Protein A-agarose beads. A commercially available immunoprecipitation kit was used for the assay. The experimental procedures were the same as described in the previous paper . Purified NFG5-hFasLECD protein was employed as the authentic positive control sample.
Cell culture and cytotoxicity assay
Human colorectal adenocarcinoma cell line HT-29 cells (catalog no. HTB-38) were purchased from ATCC, and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (Biowest), and 1 mM sodium pyruvate at 37°C in a 95% humidified air-5% CO2 incubator. Cell passages were carried at 80% confluence at a ratio of 1:3, using trypsin EDTA (Sigma) to detach cells.
HT-29 cells were seeded at 2 × 104 cells/well in 100 μl medium in 96 wells microplates (BD Falcon). Cells were allowed to attach and grow for 24 h under 37°C, 95% humidified air-5% CO2 incubation conditions before treatment with Fas ligand samples. Serially diluted Fas ligand samples (0.1, 1, 10, 100 and 1000 ng/ml) mixed with or without anti-FLAG® M2 antibody (Sigma, 2 μg/ml) were incubated for 48 h or 72 h in a final volume of 100 μl medium. The control experiments and the vehicle experiments consisted of medium only treatment and sample dilution buffer (50 mM sodium acetate plus 150 mM NaCl, pH 5.6) in medium treatment, respectively. Either of them was incubated with or without 2 μg/ml anti-FLAG® M2 antibody.
Cell viability was evaluated using MTT assay. Ten μl of MTT (5 mg/ml in PBS) was added to each well at the end of the treatment, and the plates were protected from light and incubated overnight at 37°C in a 95% humidified air-5% CO2 incubator. Then, 100 μl of 10% SDS solution was added to dissolve the formed formazan, and the plates were incubated for extra 24 h under the same conditions. Absorbance at 570 nm was recorded using Power Scan plate reader (Dainippon pharmaceuticals). Cell viability was calculated as % of control. A series of experiments were conducted in triplicate for each independent condition. Duplicate series of experimental data (n = 6 in total) were used for statistical evaluation of cell viability.
Human Fas ligand extracellular domain
Human Fas receptor extracellular domain
A fusion protein composed of human Fas receptor extracellular domain and human IgG1-Fc domain containing a thrombin cleavage site within the fusion-region sequence
Amino acid residues
- BMGY medium:
Buffered glycerol complex medium
- BMMY medium:
Buffered methanol complex medium
- EDTA Na:
Ethylenediaminetetraaceticacid sodium salt
Sodium dodecyl sulfate polyacrylamide gel-electrophoresis.
This work was supported by a grant for operating expenses from the Ministry of Economy, Trade and Industry, Japan. The author would like to thank Mr. Y. Kawabata (Fujimori Kogyo Co. Ltd.) and Mr. J. Baba (Nihon Pall Ltd.) for technical assistance with the devices used in this study. The cell culture and cytotoxicity assay was performed as a custom service of Research Institute of Biomolecule Metrology, Co. Ltd., Japan. The author thanks to Drs. A. E. Omri and Y. Sugiyama for technical assistance and helpful advice on cytotoxicity assay. Editorial assistance in the preparation of the manuscript by Prof. C.S. Langham (School of Dentistry, Nihon University) is also gratefully acknowledged.
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