BACKGROUND
abnormal apoptosis can cause many serious diseases, such as cancers and rheumatoid arthritis, in the human body
<cit> .
therefore, it has been assumed that the successful artificial control of apoptotic processes will play a substantial role in therapeutic interventions of these diseases
<cit> .
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
<cit> .
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
<cit> .
accordingly, functional recombinant wild-type human fas ligand extracellular domain  and its derivatives have been produced in heterologous systems using several kinds of expression hosts including escherichia coli <cit> , pichia pastoris <cit>  and dictyostelium discoideum <cit> .
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
<cit> .
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
<cit>  and the clarification of the origin of carbohydrate recognition specificity
<cit> .
the extracellular domain of human fas ligand locates at the carboxyl-terminal region  of whole molecule consisting of  <dig> aa, and independently exists as trimeric subunits without the help of other parts of the molecule under physiological conditions
<cit> .
this domain contains three n-glycosylation sites  and two cysteine residues  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®- <dig> tag sequence
<cit>  and the deletion of the non-essential region in trimerization
<cit>  significantly increased the secretion level of the products.
we also showed that two asparagine residues  could be mutated to glutamine residues without serious reduction of the secretion level
<cit> , and that the remaining heterogeneous n-glycan chains attached to asn  <dig> in the n-terminal flag®- <dig> tagged double n-glycosylation sites mutant could be trimmed to homogeneous n-acetyl glucosamine residues without impairing the binding activity
<cit>  toward a recombinant human fas receptor extracellular domain  derivative produced in silkworm larvae
<cit> .
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®- <dig> 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.
RESULTS
enhanced yield of tag-free hfaslecd using disposable culture-bag
figure
<dig> summarizes the system used for the secretory production of recombinant hfaslecds in this study.
as shown in figure
1a, the gene organization of the expression unit was essentially the same as in the previous study
<cit> .
figure
1b illustrates the cultivation system using a plastic culture-bag schematically.
the necessary air for the growth of p. pastoris transformant cells was supplied forcibly with a diaphragm-type vacuum pump.
the culture-bag on a stainless-steel holder was rotated in a thermostatic air incubator gently  during the cultivation period in order to avoid sedimentation of the p. pastoris cells.
the dimensions of the culture bag and the air ventilation speed were 190 mm × 190 mm × 190 mm and  <dig>  liter/min, respectively.
in table
<dig>  the purification course of tag-free hfaslecd  produced using the culture-bag system and its n-glycan trimmed derivative is presented.
the purification of tag-free hfaslecd was performed through two rounds of cation-exchange chromatography .
starting from 2300 ml of the recovered supernatant after 96 h cultivation, the final  <dig>  mg yield of highly purified tag-free hfaslecd sample  was obtained.
this purification yield  corresponded to a three-fold increase as compared with the previously reported purification yield  concerning a sample of the same quality, which was obtained from a total of 4000 ml culture supernatant produced by  <dig> rounds of the 500 ml scale cultivation using a 3000 ml volume of glass baffled culture-flask as reported previously
<cit> .
*1not determined.
*2final n-glycan untrimmed product.
*3final n-glycan trimmed product.
<cit> ; f, co-immunoprecipitated materials using hfasrecd-t-fc  mutant; m, molecular-weight markers.
“+cho” and “Δcho” indicate the migration positions of n-glycan untrimmed and n-glycan trimmed tag-free hfaslecd, respectively.
as demonstrated in the previous study concerning flag®- <dig> tagged sample
<cit> , the remaining n-glycans in tag-free hfaslecd sample in this study could also be trimmed with an endo h-type glycosidase, endo hf .
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 , 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  analysis .
receptor-mediated co-immunoprecipitation using wild-type hfasrecd-t-fc and protein a-agarose beads was used for the evaluation of the binding activity of the purified hfaslecd samples toward hfasrecd.
in this study, the binding activity of the samples was also examined using a mutant hfasrecd-t-fc, which contains double amino acid residue substitutions  to remove all possible n-glycosylation sites in the hfasrecd part .
in contrast to the presence of several discrete bands in the final purified sample of isolated wild-type hfasrecd part
<cit> , this mutant gave a single band after cleavage with bovine thrombin due to the lack of heterologous n-glycosylation .
both n-glycan untrimmed and trimmed tag-free hfaslecd samples presented clear binding activity against either wild-type hfasrecd-t-fc or the n-glycan deficient mutant .
<cit> .
lanes: m, molecular-weight size markers; a, before digestion; b and c, after digestion ; d, after removal of fc fragment by protein g-agarose column; e, after fractionation by cation-exchange chromatography.
used column: resource s 1 ml.
<cit> .
lanes: m, molecular-weight size-markers; a, nfg5-hfaslecd
<cit> ; b, tag-free hfaslecd; c, n-ethylmaleimide adduct of nfg1cg4-hfaslecd, d, sunbright® me-050ma adduct of nfg1cg4-hfaslecd; e, buffer alone.
the bands in “fasr-fc” labeled region were derived from wild-type hfasrecd-t-fc sample.
b) comparison between wild-type hfasrecd-t-fc  and hfasrecd-t-fc  mutant .
lanes: m, molecular-weight size-markers; a and b, tag-free hfaslecd; c and d, n-ethylmaleimide adduct of nfg1cg4-hfaslecd; e and f, sunbright® me-050ma adduct of nfg1cg4-hfaslecd.
the bands in “fasr-fc” labeled region were derived from hfasrecd-t-fc samples.
secretory expression of nfg1cg4-tagged hfaslecd
based on promising results from the production of tag-free hfaslecd, the secretion of nfg1cg4-hfaslecd , which should have an additional reactive cysteine residue per subunit of the trimeric molecule within the  <dig> region in n-terminal flag®- <dig> tagged hfaslecd  , was examined for the applicability of the system using a disposable culture-bag.
in figure
5a, a comparison of the secretion level of nfg1cg4-hfaslecd between the cultivation system using the baffled glass flask and that using the disposable plastic bag is shown.
as seen from the densities of the corresponding bands in sds-page analysis, the secretion level was significantly improved in the culture-bag system as compared with the baffled culture-flask system at all sampling times of every 24 h interval within the 96 h cultivation .
the subunits composing secreted nfg1cg4-hfaslecd trimers mainly existed as a couple of forms, monomer and dimer, under disulfide-bridge non-reducing conditions .
both forms of nfg1cg4-hfaslecd subunits in the culture supernatant were involved in the specific binding toward wild-type hfasrecd-t-fc protein .
the possible inter-subunit disulfide bond formed from the cysteine residues in the dimeric subunits of nfg1cg4-hfaslecd was easily cleaved with 2-mercaptoethanol contained in the reducing sample loading buffer, and single major band of the reduced sample appeared at the position of monomeric form in sds-page analysis .
<cit>  ; c, buffer alone.
upper and lower arrows indicate the same as in a).
the bands in “fasr-fc” labeled region were derived from wild-type hfasrecd-t-fc sample.
chemical modification of nfg1cg4-hfaslecd with single maleimide group containing compounds
the secreted nfg1cg4-hfaslecd was first partially purified using the same cation-exchange chromatography as described for tag-free hfaslecd, and then treated with moderate concentration of tris-phosphine  under neutral ph at room temperature.
this tcep reduced product was immediately subjected to the chemical modification reaction with two kinds of single maleimide-group containing compounds, i. e. n-ethylmaleimide and sunbright® me-050ma.
sunbright® me-050ma is a mono-functional linear methoxy polyethylene glycol , which has the molecular-weight of approximately 5 kda and contains an active maleimide group at one end .
in figure
6b, sds-page analysis of the reaction mixtures at each individual step is shown.
after the treatment of partially purified nfg1cg4-hfaslecd  with 20 mm tcep for 1 h at room temperature, the possible inter-subunit disulfide bond was completely cleaved .
this disulfide bond was slightly regenerated during the desalting step to remove excess amount of tcep .
however, the original inter-subunit disulfide bond was not observed in the reaction mixture with an excess amount of single maleimide-group containing compounds .
the purification of the nfg1cg4-hfaslecd modified with single maleimide-group containing compounds was performed by cation-exchange column chromatography .
the less sharp peak following the main peak observed in the sample before the conjugation reaction  virtually disappeared from the sample after modifications.
the sodium chloride  concentrations required for elution of the chemically modified products depended on the molecular structure of the maleimide reagent.
the main peak of n-ethylmaleimide conjugated nfg1cg4-hfaslecd was eluted under almost the same nacl concentration  as that for the main peak of the sample before the conjugation reaction .
the main peak of sunbright® me-050ma conjugated nfg1cg4-hfaslecd was eluted under significantly lower nacl concentration , suggesting weaker binding to a negatively charged column carrier due to the reduced ionic interactions caused by the possible shielding effect of the attached peg moieties.
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 .
either sample presented a symmetrical single peak, suggesting the uniformity of molecular-weight of the fractionated products .
the peak retention time of the n-ethylmaleimide conjugated sample  was between the size-markers of 44 kda  and 158 kda  , and was nearly the same as observed for tag-free hfaslecd  under the identical elution condition using the same chromatography column .
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  and the peak retention time was even significantly earlier than the 158 kda size-marker .
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 .
figure
7c shows the sds-page analysis of the fractionated samples.
as shown in lane b, the n-ethylmaleimide conjugated nfg1cg4-hfaslecd sample exhibited a single band at the position of approximately 21 kda.
on the other hand, the sunbright® me-050ma conjugated nfg1cg4-hfaslecd sample exhibited two bands, which consisted of one major thick band migrated at approximately 30 kda and another minor faint band at 21 kda .
this result suggested that greatest portions of free cysteine residues in the nfg1cg4-hfaslecd sample were conjugated with sunbright® me-050ma moieties.
table
<dig> summarizes the purification course of nfg1cg4-hfaslecd conjugated with the single maleimide group containing compounds.
starting from 2500 ml of the culture supernatant,  <dig>  mg and  <dig>  mg of the purified products were recovered with respect to the n-ethylmaleimide adduct and the sunbright® me-050ma adduct, respectively.
*1not determined.
*2normalized to the values starting from initial sample volume .
*3final n-ethylmaleimide conjugated product.
*4final sunbright® me-050-ma conjugated product.
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 .
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 , 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 .
cross-linking of nfg1cg4-hfaslecd with two maleimide-groups containing polyethylene glycol
partially purified nfg1cg4-hfaslecd reduced with moderated concentration of tcep under neutral ph at room temperature was also examined for cross-linking with two maleimide groups containing compound, sunbright® de-100ma.
sunbright® de-100ma is a homo-bifunctional linear peg with the molecular-weight of approximately 10 kda, which has two maleimide groups at either end of the molecule .
in figure
8b, an elution profile of the reaction mixture after the cross-linking reaction on a size-exclusion chromatography column and sds-page analysis of the fractionated peaks in the size-exclusion chromatography are displayed.
peaks, i, ii and iii contained the cross-linked materials.
the band with the highest molecular-weight between molecular size-markers of  <dig>  kda and  <dig>  kda found in the peak i sample  was regarded to correspond to the major impurity protein found in the sample before the cross-linking reaction .
from the molecular-weight of the bands, the upper arrowed band and lower arrowed band found in peaks i, ii and iii  were considered to be two nfg1cg4-hfaslecd subunits cross-linked by one sunbright® de-100ma molecule and one nfg1cg4-hfaslecd subunit conjugated with one sunbright® de-100ma molecule, respectively.
the purification yields of the chemically modified products contained in peaks ii and iii were  <dig>  mg and  <dig>  mg, respectively, which were obtained from 2500 ml of the culture supernatant of nfg1cg4-hfaslecd.
the samples fractionated as peaks ii and iii were independently re-chromatographed on the same column again .
both peaks held approximately the same original retention time, suggesting that the product in peak ii was not just a transient aggregation of the product in peak iii.
the same amounts of purified proteins were subjected to sds-page analysis .
this revealed that peak ii and peak iii contained essentially the same subunit components with identical compositions.
materials in peak ii were considered as an additional associated product of materials in peak iii, which were composed of more highly cross-linked nfg1cg4-hfaslecd.
all components in peaks ii and iii were found in the precipitated samples obtained in the receptor-mediated co-immunoprecipitaion experiments using either wild-type or the mutant hfasrecd-t-fc , which suggested that the cross-linked products in peaks ii and iii retained the binding activity toward hfasrecd.
cytotoxic activity of chemically modified nfg1cg4-hfaslecd
in order to examine the cytotoxic activity of nfg1cg4-hfaslecd containing site-specific modification with a maleimide compound, the cell viability of a human colorectal adenocarcinoma ht- <dig> cell line was evaluated using 3- assay.
the susceptibility enhancement in apoptosis induction by pretreatment with interferon-γ concerning this cell line
<cit>  was not applied here.
figure
10a shows the morphologic change of ht- <dig> cells induced by treatment with n-ethylmaleimide conjugated nfg1cg4-hfaslecd, indicating the occurrence of apoptosis.
a number of apparently dark and shrank cells suggesting the occurrence of apoptosis, which were not present in the sample at 72 h treatment by buffer alone , were observed in the micrograph of the sample at 72 h treatment by 100 ng/ml of n-ethylmaleimide conjugated nfg1cg4-hfaslecd .
in figure
10b, the cytotoxic effect of the purified n-ethylmaleimide conjugated nfg1cg4-hfaslecd on ht- <dig> cells in the presence or absence of anti-flag® m <dig> antibody is shown.
the fas ligand sample exhibited significant cell-death inducing activity in a dose and time dependent manner in the presence of the m <dig> antibody.
the buffer alone sample in vehicle experiment showed no significant activity even after 72 h treatment under either condition, and the cell viability  in the presence and absence of the m <dig> antibody was  <dig>  ±  <dig> % and 103 ±  <dig> % of control, respectively.
the above results suggested the induction of apoptosis via fas ligation by n-ethylmaleimide conjugated nfg1cg4-hfaslecd was evident only after cross-linking through the recognition of flag® sequence existing in the nfg1cg4-tag by m <dig> antibody.
discussion
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
<cit> .
a methanol utilization plus strain, p. pastoris gs <dig> , has been used as the host in the secretory production of this protein
<cit> .
the best purification yield of  <dig>  mg/l was reported with the nfg5-hfaslecd  containing the mutations of n184q and n250q
<cit> .
however, the deletion of nfg <dig> tag sequence in this mutant resulted in a large reduction of the purification yield to  <dig>  mg/l
<cit> .
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 .
in another experiment for the production of nfg1cg4-hfaslecd in p. pastoris gs <dig> strain using 4000 ml bmmy medium in a  <dig> liter volume plastic culture-bag , 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 .
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
<cit> .
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
<cit> .
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
<cit>  and the three-dimensional structures produced by in silico modeling
<cit> .
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
<cit> .
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
<cit> .
nfg5-hfaslecd  is considered to contain a single disulfide bond between cys  <dig> and cys  <dig> within a structurally buried region by analogy with the available three-dimensional structure of hfaslecd
<cit> .
however, it is known that tcep can selectively reduce solvent exposed disulfide bonds under mild reaction conditions without affecting the structurally buried one
<cit> .
therefore, the n-terminal tag sequence region in nfg5-hfaslecd
<cit>  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
<cit> .
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 <dig>  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
<cit> .
the n-ethylmaleimide conjugated nfg1cg4-hfaslecd exhibited a cell-death inducing activity against ht- <dig> cells, a human colorectal adenocarcinoma cell line.
the cytotoxic activity was significant only after cross-linking by m <dig> antibody specific for flag®-tag as depicted with the activity of a flag®-tagged trimeric hfaslecd against jurkat, raji and hela cells
<cit> .
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 <cit> .
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
<cit> .
this also showed that the two n-glycan chains at asn <dig> and asn <dig> 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 dcr <dig> with a similar affinity to hfasrecd at the same time
<cit> , which can be one of the possible reasons leading to onset of diseases caused by excessive cellular proliferation
<cit> .
overexpression of dcr <dig> decoy receptor was found in several types of cancers and autoimmune diseases
<cit> .
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 acrp <dig> collagen domain region, displayed much higher killing activity than trimeric hfaslecd against several kinds of tumor cells
<cit> .
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
<cit>  by conjugations with either fluorescent dyes
<cit>  or luminescent proteins
<cit> .
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
<cit> , prognosis
<cit> , response to drug treatment
<cit>  and mortality
<cit>  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.
CONCLUSIONS
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.

 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. 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. accordingly, functional recombinant wild-type human fas ligand extracellular domain and its derivatives have been produced in heterologous systems using several kinds of expression hosts including escherichia coli, pichia pastoris 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 of whole molecule consisting of 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 and two cysteine residues 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®- tag sequence and the deletion of the non-essential region in trimerization significantly increased the secretion level of the products. we also showed that two asparagine residues could be mutated to glutamine residues without serious reduction of the secretion level, and that the remaining heterogeneous n-glycan chains attached to asn in the n-terminal flag®- 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 derivative produced in silkworm larvae. 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®- 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 figure  summarizes the system used for the secretory production of recombinant hfaslecds in this study. as shown in figure  1a, the gene organization of the expression unit was essentially the same as in the previous study. figure  1b illustrates the cultivation system using a plastic culture-bag schematically. the necessary air for the growth of p. pastoris transformant cells was supplied forcibly with a diaphragm-type vacuum pump. the culture-bag on a stainless-steel holder was rotated in a thermostatic air incubator gently during the cultivation period in order to avoid sedimentation of the p. pastoris cells. the dimensions of the culture bag and the air ventilation speed were 190 mm × 190 mm × 190 mm and liter/min, respectively. in table  the purification course of tag-free hfaslecd produced using the culture-bag system and its n-glycan trimmed derivative is presented. the purification of tag-free hfaslecd was performed through two rounds of cation-exchange chromatography. starting from 2300 ml of the recovered supernatant after 96 h cultivation, the final  mg yield of highly purified tag-free hfaslecd sample was obtained. this purification yield corresponded to a three-fold increase as compared with the previously reported purification yield concerning a sample of the same quality, which was obtained from a total of 4000 ml culture supernatant produced by rounds of the 500 ml scale cultivation using a 3000 ml volume of glass baffled culture-flask as reported previously. *1not determined. *2final n-glycan untrimmed product. *3final n-glycan trimmed product.; f, co-immunoprecipitated materials using hfasrecd-t-fc mutant; m, molecular-weight markers. “+cho” and “Δcho” indicate the migration positions of n-glycan untrimmed and n-glycan trimmed tag-free hfaslecd, respectively. as demonstrated in the previous study concerning flag®- 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. 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, 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 analysis. receptor-mediated co-immunoprecipitation using wild-type hfasrecd-t-fc and protein a-agarose beads was used for the evaluation of the binding activity of the purified hfaslecd samples toward hfasrecd. in this study, the binding activity of the samples was also examined using a mutant hfasrecd-t-fc, which contains double amino acid residue substitutions to remove all possible n-glycosylation sites in the hfasrecd part. in contrast to the presence of several discrete bands in the final purified sample of isolated wild-type hfasrecd part, this mutant gave a single band after cleavage with bovine thrombin due to the lack of heterologous n-glycosylation. both n-glycan untrimmed and trimmed tag-free hfaslecd samples presented clear binding activity against either wild-type hfasrecd-t-fc or the n-glycan deficient mutant.. lanes: m, molecular-weight size markers; a, before digestion; b and c, after digestion; d, after removal of fc fragment by protein g-agarose column; e, after fractionation by cation-exchange chromatography. used column: resource s 1 ml.. lanes: m, molecular-weight size-markers; a, nfg5-hfaslecd; b, tag-free hfaslecd; c, n-ethylmaleimide adduct of nfg1cg4-hfaslecd, d, sunbright® me-050ma adduct of nfg1cg4-hfaslecd; e, buffer alone. the bands in “fasr-fc” labeled region were derived from wild-type hfasrecd-t-fc sample. b) comparison between wild-type hfasrecd-t-fc and hfasrecd-t-fc mutant. lanes: m, molecular-weight size-markers; a and b, tag-free hfaslecd; c and d, n-ethylmaleimide adduct of nfg1cg4-hfaslecd; e and f, sunbright® me-050ma adduct of nfg1cg4-hfaslecd. the bands in “fasr-fc” labeled region were derived from hfasrecd-t-fc samples. secretory expression of nfg1cg4-tagged hfaslecd based on promising results from the production of tag-free hfaslecd, the secretion of nfg1cg4-hfaslecd, which should have an additional reactive cysteine residue per subunit of the trimeric molecule within the region in n-terminal flag®- tagged hfaslecd, was examined for the applicability of the system using a disposable culture-bag. in figure  5a, a comparison of the secretion level of nfg1cg4-hfaslecd between the cultivation system using the baffled glass flask and that using the disposable plastic bag is shown. as seen from the densities of the corresponding bands in sds-page analysis, the secretion level was significantly improved in the culture-bag system as compared with the baffled culture-flask system at all sampling times of every 24 h interval within the 96 h cultivation. the subunits composing secreted nfg1cg4-hfaslecd trimers mainly existed as a couple of forms, monomer and dimer, under disulfide-bridge non-reducing conditions. both forms of nfg1cg4-hfaslecd subunits in the culture supernatant were involved in the specific binding toward wild-type hfasrecd-t-fc protein. the possible inter-subunit disulfide bond formed from the cysteine residues in the dimeric subunits of nfg1cg4-hfaslecd was easily cleaved with 2-mercaptoethanol contained in the reducing sample loading buffer, and single major band of the reduced sample appeared at the position of monomeric form in sds-page analysis.; c, buffer alone. upper and lower arrows indicate the same as in a). the bands in “fasr-fc” labeled region were derived from wild-type hfasrecd-t-fc sample. chemical modification of nfg1cg4-hfaslecd with single maleimide group containing compounds the secreted nfg1cg4-hfaslecd was first partially purified using the same cation-exchange chromatography as described for tag-free hfaslecd, and then treated with moderate concentration of tris-phosphine under neutral ph at room temperature. this tcep reduced product was immediately subjected to the chemical modification reaction with two kinds of single maleimide-group containing compounds, i. e. n-ethylmaleimide and sunbright® me-050ma. sunbright® me-050ma is a mono-functional linear methoxy polyethylene glycol, which has the molecular-weight of approximately 5 kda and contains an active maleimide group at one end. in figure  6b, sds-page analysis of the reaction mixtures at each individual step is shown. after the treatment of partially purified nfg1cg4-hfaslecd with 20 mm tcep for 1 h at room temperature, the possible inter-subunit disulfide bond was completely cleaved. this disulfide bond was slightly regenerated during the desalting step to remove excess amount of tcep. however, the original inter-subunit disulfide bond was not observed in the reaction mixture with an excess amount of single maleimide-group containing compounds. the purification of the nfg1cg4-hfaslecd modified with single maleimide-group containing compounds was performed by cation-exchange column chromatography. the less sharp peak following the main peak observed in the sample before the conjugation reaction virtually disappeared from the sample after modifications. the sodium chloride concentrations required for elution of the chemically modified products depended on the molecular structure of the maleimide reagent. the main peak of n-ethylmaleimide conjugated nfg1cg4-hfaslecd was eluted under almost the same nacl concentration as that for the main peak of the sample before the conjugation reaction. the main peak of sunbright® me-050ma conjugated nfg1cg4-hfaslecd was eluted under significantly lower nacl concentration, suggesting weaker binding to a negatively charged column carrier due to the reduced ionic interactions caused by the possible shielding effect of the attached peg moieties. 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. either sample presented a symmetrical single peak, suggesting the uniformity of molecular-weight of the fractionated products. the peak retention time of the n-ethylmaleimide conjugated sample was between the size-markers of 44 kda and 158 kda, and was nearly the same as observed for tag-free hfaslecd under the identical elution condition using the same chromatography column. 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 and the peak retention time was even significantly earlier than the 158 kda size-marker. 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. figure  7c shows the sds-page analysis of the fractionated samples. as shown in lane b, the n-ethylmaleimide conjugated nfg1cg4-hfaslecd sample exhibited a single band at the position of approximately 21 kda. on the other hand, the sunbright® me-050ma conjugated nfg1cg4-hfaslecd sample exhibited two bands, which consisted of one major thick band migrated at approximately 30 kda and another minor faint band at 21 kda. this result suggested that greatest portions of free cysteine residues in the nfg1cg4-hfaslecd sample were conjugated with sunbright® me-050ma moieties. table  summarizes the purification course of nfg1cg4-hfaslecd conjugated with the single maleimide group containing compounds. starting from 2500 ml of the culture supernatant,  mg and  mg of the purified products were recovered with respect to the n-ethylmaleimide adduct and the sunbright® me-050ma adduct, respectively. *1not determined. *2normalized to the values starting from initial sample volume. *3final n-ethylmaleimide conjugated product. *4final sunbright® me-050-ma conjugated product. 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. 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, 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. cross-linking of nfg1cg4-hfaslecd with two maleimide-groups containing polyethylene glycol partially purified nfg1cg4-hfaslecd reduced with moderated concentration of tcep under neutral ph at room temperature was also examined for cross-linking with two maleimide groups containing compound, sunbright® de-100ma. sunbright® de-100ma is a homo-bifunctional linear peg with the molecular-weight of approximately 10 kda, which has two maleimide groups at either end of the molecule. in figure  8b, an elution profile of the reaction mixture after the cross-linking reaction on a size-exclusion chromatography column and sds-page analysis of the fractionated peaks in the size-exclusion chromatography are displayed. peaks, i, ii and iii contained the cross-linked materials. the band with the highest molecular-weight between molecular size-markers of  kda and  kda found in the peak i sample was regarded to correspond to the major impurity protein found in the sample before the cross-linking reaction. from the molecular-weight of the bands, the upper arrowed band and lower arrowed band found in peaks i, ii and iii were considered to be two nfg1cg4-hfaslecd subunits cross-linked by one sunbright® de-100ma molecule and one nfg1cg4-hfaslecd subunit conjugated with one sunbright® de-100ma molecule, respectively. the purification yields of the chemically modified products contained in peaks ii and iii were  mg and  mg, respectively, which were obtained from 2500 ml of the culture supernatant of nfg1cg4-hfaslecd. the samples fractionated as peaks ii and iii were independently re-chromatographed on the same column again. both peaks held approximately the same original retention time, suggesting that the product in peak ii was not just a transient aggregation of the product in peak iii. the same amounts of purified proteins were subjected to sds-page analysis. this revealed that peak ii and peak iii contained essentially the same subunit components with identical compositions. materials in peak ii were considered as an additional associated product of materials in peak iii, which were composed of more highly cross-linked nfg1cg4-hfaslecd. all components in peaks ii and iii were found in the precipitated samples obtained in the receptor-mediated co-immunoprecipitaion experiments using either wild-type or the mutant hfasrecd-t-fc, which suggested that the cross-linked products in peaks ii and iii retained the binding activity toward hfasrecd. cytotoxic activity of chemically modified nfg1cg4-hfaslecd in order to examine the cytotoxic activity of nfg1cg4-hfaslecd containing site-specific modification with a maleimide compound, the cell viability of a human colorectal adenocarcinoma ht- cell line was evaluated using 3- assay. the susceptibility enhancement in apoptosis induction by pretreatment with interferon-γ concerning this cell line was not applied here. figure  10a shows the morphologic change of ht- cells induced by treatment with n-ethylmaleimide conjugated nfg1cg4-hfaslecd, indicating the occurrence of apoptosis. a number of apparently dark and shrank cells suggesting the occurrence of apoptosis, which were not present in the sample at 72 h treatment by buffer alone, were observed in the micrograph of the sample at 72 h treatment by 100 ng/ml of n-ethylmaleimide conjugated nfg1cg4-hfaslecd. in figure  10b, the cytotoxic effect of the purified n-ethylmaleimide conjugated nfg1cg4-hfaslecd on ht- cells in the presence or absence of anti-flag® m antibody is shown. the fas ligand sample exhibited significant cell-death inducing activity in a dose and time dependent manner in the presence of the m antibody. the buffer alone sample in vehicle experiment showed no significant activity even after 72 h treatment under either condition, and the cell viability in the presence and absence of the m antibody was  ±  % and 103 ±  % of control, respectively. the above results suggested the induction of apoptosis via fas ligation by n-ethylmaleimide conjugated nfg1cg4-hfaslecd was evident only after cross-linking through the recognition of flag® sequence existing in the nfg1cg4-tag by m antibody. discussion 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 gs, has been used as the host in the secretory production of this protein. the best purification yield of  mg/l was reported with the nfg5-hfaslecd containing the mutations of n184q and n250q. however, the deletion of nfg tag sequence in this mutant resulted in a large reduction of the purification yield to  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. in another experiment for the production of nfg1cg4-hfaslecd in p. pastoris gs strain using 4000 ml bmmy medium in a liter volume plastic culture-bag, 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. 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 and the three-dimensional structures produced by in silico modeling. 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 is considered to contain a single disulfide bond between cys and cys 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. 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 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. the n-ethylmaleimide conjugated nfg1cg4-hfaslecd exhibited a cell-death inducing activity against ht- cells, a human colorectal adenocarcinoma cell line. the cytotoxic activity was significant only after cross-linking by m 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. this also showed that the two n-glycan chains at asn and asn 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 dcr 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 dcr 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 acrp 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, 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. 
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. 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. 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. 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. human fas ligand extracellular domain is an agonistic death ligand protein that has potential applications for medical purposes. 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 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. site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. 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.
 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 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- 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. human fas ligandextracellular domainproductionpichia pastorisyield enhancementsite-specific chemical modification 