BACKGROUND
aminoacyl-trna synthetases  are enzymes that catalyze the essential first step of protein synthesis by covalently attaching each of the  <dig> standard amino acids to their cognate trna.
because the reactions require the capacity to recognize trnas as well as small chemicals such as amino acids and atp, the structures of aarss are well equipped for interacting with diverse molecules that may be associated with their functional versatility  <cit> .
aarss, such as glutamyl-prolyl-trna synthetase, glutaminyl-trna synthetase, and lysyl-trna synthetase, are now known to be multifunctional, participating in a variety of functions including transcription, translation, splicing, inflammation, thrombopoiesis, and apoptosis  <cit> .
human tyrosyl-trna synthetase  was the first example of a trna synthetase that played a role in cell signaling and thrombopoiesis.
it has been demonstrated that human full-length tyrrs has no known cytokine activity  <cit> .
however, proteolytic removal of the c-domain from tyrrs activates its cytokine function  <cit> .
the n-terminal fragment, named mini-tyrrs, specifically stimulates migration of endothelial cells and polymorphonuclear leukocytes  <cit> .
mini-tyrrs has also been shown to activate thrombocytopoiesis in a chick chorioallantoic membrane thrombopoiesis assay  <cit> .
as in some cxc chemokines, such as il- <dig>  an glu-leu-arg motif embedded in the sequence of mini-tyrrs was shown by mutational analysis to be critical for its cytokine activity  <cit> .
although there is no crystal structure reported for full-length tyrrs, high-resolution crystal structures of mini-tyrrs and of the c-domain enabled reconstruction of the full-length enzyme, and has provided the model and structural basis for its cytokine activity  <cit> .
it is predicted that the c-terminus of the full-length protein is tethered to the n-terminal region, thus preventing elr substrate recognition  <cit> .
recombinant human tyrrs  is a full-length human tyrosyl-trna synthetase with a mutation at y341a that blocks the h-bond between the elr motif and y <dig>  as a result, the elr motif is exposed, and mini-rhtyrrs-like activity can then promote thrombocytopoiesis  <cit> .
as a potential thrombocytopoietic drug, large-scale expression and purification of rhtyrrs would be desirable.
preclinical testing involving in vitro and animal studies could then be carried out to evaluate its toxic and pharmacologic effects.
in this study, rhtyrrs was expressed at a high level in e. coli and purified for future preclinical testing.
methods
cells and antibodies
the competent e. coli strain bl <dig>  gal dcm; providded by atyr pharma) was used as the host for rhtyrrs expression.
this strain was transformed with the pet24a inducible expression vector in which the his-tag sequence was deleted and the t <dig> promoter was replaced with a tac promoter.
a mouse anti-human il- <dig> monoclonal antibody , rabbit anti-human il- <dig> polyclonal antibody , and goat anti-rabbit igg-hrp sc- <dig> antibody  were used.
growth conditions
a clone with a high rhtyrrs expression level was cultivated in lb agar and m9ca medium .
trace element solution  consisted of  <dig>  g feso4 · 7h2o, 2 g mncl2 · 4h2o,  <dig>  g coso4 · 7h2o,  <dig>  g cacl2 · 2h2o,  <dig>  g cucl2 · 2h2o, and  <dig>  g znso4 · 7h2o.
feeding solutions were 50% glycerol, 250 g/l glucose, and 100 g/l yeast extract.
the glucose and mgso <dig> solutions were sterilized separately.
kanamycin sulfate was added to a final concentration of 100 μg/ml in both the m9ca and feeding solutions.
fermentation was performed in a 5-l bioflo  <dig> fermenter  with automated control of: the ph at  <dig>  by the addition of ammonium hydroxide, dissolved o <dig> at 70% by providing pure oxygen, and agitation at 700 rpm.
air was provided at a flow rate of  <dig>  l/min and the temperature was controlled at 30°c.
fermentation was conducted according to the process developed by shiloach et al.
<cit> .
the batch phase ends when cells have used up the available glucose.
the best indications that the batch phase has ended include a sharp decrease in stirrer speed and an increase in po <dig>  the bacterial concentration was measured off-line by the optical density at 600 nm and induced with  <dig>  mm isopropylthio-β-galactoside  once it reached an od <dig> of  <dig> .
after 6 h of  <dig>  mm iptg induction, the cells were harvested by centrifugation at  <dig>  × g for 30 min.
the cell pellet was stored at -70°c.
cell lysis
harvested cell pellets were resuspended in  <dig> volumes of 20 mmol/l hac-naac buffer  and subjected to two cycles of microfluidization at 1000 bar.
the crude extract was then centrifuged at  <dig>  × g for 60 min.
cation exchange chromatography
the clarified supernatant was loaded onto a sp sepharose fast flow column  that was pre-equilibrated with 20 mmol/l hac-naac buffer .
the bound proteins were eluted with a linear nacl gradient .
fractions containing rhtyrrs were pooled and analyzed by sds-page.
gel filtration
the pooled fractions were loaded onto a sephadex-g <dig> column  pre-equilibrated with 20 mmol/l phosphate buffer  and rhtyrrs was washed with 20 mmol/l pb at 5 ml/min.
fractions were collected from the column and analyzed by sds-page.
anion exchange chromatography
the diluted product solution was loaded onto a 50-ml q sepharose fast flow column  pre-equilibrated with 20 mmol/l pb .
rhtyrrs was eluted with a linear nacl gradient  and identified by sds-page via coomassie blue staining.
sds-page and sequencing of the n-terminal amino acids
electrophoresis was carried out in 1-mm-thick gels using biorad minigel apparatus.
coomassie staining was performed as previously described, except that microwave heating was used at each staining step to reduce the total staining and destaining procedure time to 30 min.
the protein concentration was measured using bsa as a standard.
the sequence of the n-terminal amino acids of purified rhtyrrs was determined using a protein sequencer .
western blotting
proteins resolved in a pre-cast bis-tris gel  were electrotransferred to a pvdf membrane followed by blocking in 10% bsa solution prepared in tbst .
the membrane was then incubated with anti-rhtyrrs monoclonal antibody  for 90 min at room temperature.
after washing, the membrane was incubated with peroxidase-conjugated goat anti-mouse igg  for 60 min at room temperature.
all antibody incubations and washing steps were carried out in tbst.
the immunoreactive bands were visualized with a western blot kit .
reversed-phase hplc
reversed-phase hplc was used to study the purity of rhtyrrs.
a higgins proto  <dig> c <dig> column  was used at a flow rate of 1 ml/min and at a temperature of 40°c.
the column was eluted with a linear 30-min gradient from 5% b to 95% b  in hplc water; b =  <dig> % tfa in acetonitrile) and monitored by absorbance at 215 nm with a total run time of 40 min.
mass spectrometry analysis
the purified protein was analyzed by mass spectrometry.
molecular weight measurements were made by lc-ms with a quadrupole-time-of-flight mass spectrometer at the fudan university institute of biomedical science.
aminoacylation assay
aminoacylation activity was determined at ambient temperature in 150 mmol/l tris–hcl , 150 mmol/l kcl, 10 mmol/l mgcl <dig>  10 mmol/l β-mercaptoethanol, 4 mmol/l atp, and 10 mmol/l tyrosine .
human trnatyr was prepared as previously described  <cit> .
before each assay, 100 mmol/l human trnatyr was annealed by heating at 65°c for 5 min and cooled to room temperature.
reactions were initiated by the addition of rhtyrrs  to the reaction mixture.
aliquots were taken at fixed intervals and spotted onto whatman filter discs saturated with 5% trichloroacetic acid and dried.
the filters were placed into ice-cold 5% trichloroacetic acid and washed three times with fresh 5% trichloroacetic acid at 4°c and once with 95% ethanol.
the level of trna aminoacylation was then quantitated by liquid scintillation counting.
biological activity assay
rhtyrrs activity was tested using elisa to measure il- <dig> secretion by rhtyrrs-treated hl- <dig> cells.
cells were thawed from vials and cultured according to the american type culture collection instructions, except that rpmi- <dig>  was supplemented with 10% heat-inactivated fetal bovine serum  and used as growth medium.
cells were plated in a 96-well plate at a density of 1 ×  <dig> cells per 1 ml of medium on the day of the assay.
the rhtyrrs was added immediately after cell plating.
cells were plated at 108 μl- <dig> per well.
the protein was diluted in sterile phosphate-buffered saline  to a concentration of 10 μmol/l .
the 10× protein stock was serially diluted and 12 μl was added to each well such that the final protein concentrations were 500 nmol/l, 250 nmol/l, 125 nmol/l,  <dig>  nmol/l,  <dig>  nmol/l, and  <dig>  nmol/l.
pbs was used as a control.
cells were incubated at 37°c and 5% co <dig> for 24 h. the 96-well plate was then spun for 10 min at room temperature in a swinging bucket centrifuge at  <dig>  × g. the plate was tilted at a 45° angle and 100 μl of media per well was transferred to a new 96-well plate.
the amount of secreted il- <dig> protein in the media was then determined by sandwich elisa.
RESULTS
rhtyrrs expression and purification
to evaluate the long-term stability of the plasmid/strain combination, bacteria were first grown in shake flasks for  <dig> generations by repeated dilution before the analysis of rhtyrrs expression.
a typical growth curve is shown in figure 1a.
sds-page analysis of expressed rhtyrrs is shown in figure 1b.
the expression level after  <dig> generations was comparable to that produced by the cells after five generations.
approximately  <dig> – <dig>  g/l of rhtyrrs was produced under these growth conditions.
when rhtyrrs was expressed in e. coli strain bl <dig> using m9ca medium and fed-batch methodology, approximately 50% of the protein was soluble.after 6 h of iptg induction, cells were harvested by centrifugation and lysed by two cycles of microfluidization.
the concentrated media were subjected to cation exchange, gel filtration, and anion exchange chromatography, yielding rhtyrrs of up to 97% purity.
rhtyrrs was eluted with  <dig>  mol/l nacl-pb via cation exchange, then subjected to gel filtration to exchange the buffer to pb; all pooled fractions were then loaded onto a 50 ml q sepharose fast flow column pre-equilibrated with 20 mmol/l pb .
rhtyrrs was eluted with less than  <dig>  mol/l nacl-pb during anion exchange.
around  <dig>  g of purified rhtyrrs was generated from 3 l of culture.
the products from the optimized rhtyrrs purification process, consisting of cell lysis followed by cation exchange chromatography, gel filtration, and anion exchange chromatography, are shown in figure  <dig>
rhtyrrs characterization
the identity of rhtyrrs was verified by edman degradation to compare the n-terminal sequence of rhtyrrs to the expected mini-tyrrs sequence.
up to  <dig> amino acids from the n-terminus of rhtyrrs were consistently identical to the reference sequence: met-gly-asp-ala-pro-ser-pro-glu-glu-lys-leu-his-leu-ile-trp.
we also sought to confirm the identity of rhtyrrs by resolving the sample  in parallel with a reference standard by sds-page followed by western blotting using a polyclonal antibody .
reversed-phase hplc
a reversed-phase hplc was developed as a purity indication assay.
figure  <dig> shows the analysis of rhtyrrs purified using chromatography steps.
this method will be transferred to the contract manufacturer organization or its approved vendor, as appropriate.
the retention time of rhtyrrs in the c <dig> column was  <dig>  min with purity up to 97%.
mass spectrometry analysis
an accurate mass can be detected by intact mass analysis using lc-ms. peaks corresponding to various protonated rhtyrrs species are marked.
mass analysis determined that rhtyrrs has a molecular weight of  <dig>  kd .
this is identical to that identified by sds-page.
aminoacylation assay
to test whether rhtyrrs retained the ability to aminoacylate its cognate trna molecule, we took advantage of the inherent synthetase activity of rhtyrrs to verify proper protein folding and assess biological activity during the expression and purification process.
the incorporation of 3h tyrosine into trna is a well-established and reproducible assay  <cit>  .
biological activity assay
we used a sandwich elisa to test il- <dig> released by hl- <dig> cells in the presence of rhtyrrs.
the standard il- <dig> concentrations used for elisa were 1200 ng/ml, 600 ng/ml, 300 ng/ml, 150 ng/ml, 75 ng/ml, and  <dig>  ng/ml as shown in figure 6a, and r <dig> was  <dig> .
figure 6b shows the amounts of il- <dig> released by hl- <dig> cells incubated in the presence of rhtyrrs at concentrations between  <dig>  nmol/l and 1000 nmol/l, based on the standard il- <dig> shown in figure 6a.
discussion and
CONCLUSIONS
to facilitate the use of rhtyrrs proteins in regulating thrombopoietic activity and to enable structural characterization of this protein, we established a system for the production of large amounts of rhtyrrs.
a purity of approximately 97% was routinely achieved with <2% individual contaminants and little to no aggregation.
the final, optimized process consists of cell lysis followed by a sequence of three chromatography steps.
the first step  captures rhtyrrs while contaminants flow through, the second step  exchanges the buffer to pb, and the last step  removes the remaining process-related impurities.
the final concentration is 1 mg/ml in pbs .
preliminary characterization of rhtyrrs was performed using western blotting, hplc, mass spectrometry, and n-terminal sequencing to verify the identity of the protein.
the ability of the purified rhtyrrs to aminoacylate its cognate trna molecule was also tested, and analysis of the inherent synthetase activity of rhtyrrs was performed to verify proper protein folding and assess biological activity during the expression and purification processes.
kinetic parameters from different preparations of rhtyrrs will be compared with a well-characterized reference standard.
purification lots that are within the established specifications will be deemed acceptable for release.
this assay is still under development but a representative example of the linearity of product accumulation over time is shown below with a range of rhtyrrs concentrations.
the y341a substitution activated full-length tyrrs for stimulation of cell proliferation.
similar results were observed in an endothelial cell migration assay.
in these experiments, wounds were created in monolayers of human umbilical vein endothelial cells  <cit> .
we predict that rhtyrrs not only has il-8-like cytokine activity, but also can enhance il- <dig> production from monocytes  <cit> .
the biological assays of hl- <dig> stimulation with rhtyrrs presented here demonstrate that this may be the case.
the findings presented here will be important for future preclinical studies.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
yl, lz and xz performed the protein expression and purification; yz and zf analyzed the protein activity; yl and wm designed the experiments, analyzed data, and wrote the paper; my supervised the experiments and designed the study.
all authors read and approved the final manuscript.
authors’ information
yongjiang lang and yanling zhang are first author.
acknowledgments
we thank atyr pharma for providing the plasmid, e. coli strain, and the antibody for rhtyrrs.
we really appreciate the advise of dr. xianglei yang and dr. paul schimmel from the scripps research institute.
this study was supported by the national natural science foundation of china , and science and technology commission of shanghai municipality .
this study was also supported by the state key laboratory of biotherapy/collaborative innovation center for biotherapy, west china hospital, sichuan university.

 aminoacyl-trna synthetases are enzymes that catalyze the essential first step of protein synthesis by covalently attaching each of the standard amino acids to their cognate trna. because the reactions require the capacity to recognize trnas as well as small chemicals such as amino acids and atp, the structures of aarss are well equipped for interacting with diverse molecules that may be associated with their functional versatility. aarss, such as glutamyl-prolyl-trna synthetase, glutaminyl-trna synthetase, and lysyl-trna synthetase, are now known to be multifunctional, participating in a variety of functions including transcription, translation, splicing, inflammation, thrombopoiesis, and apoptosis. human tyrosyl-trna synthetase was the first example of a trna synthetase that played a role in cell signaling and thrombopoiesis. it has been demonstrated that human full-length tyrrs has no known cytokine activity. however, proteolytic removal of the c-domain from tyrrs activates its cytokine function. the n-terminal fragment, named mini-tyrrs, specifically stimulates migration of endothelial cells and polymorphonuclear leukocytes. mini-tyrrs has also been shown to activate thrombocytopoiesis in a chick chorioallantoic membrane thrombopoiesis assay. as in some cxc chemokines, such as il- an glu-leu-arg motif embedded in the sequence of mini-tyrrs was shown by mutational analysis to be critical for its cytokine activity. although there is no crystal structure reported for full-length tyrrs, high-resolution crystal structures of mini-tyrrs and of the c-domain enabled reconstruction of the full-length enzyme, and has provided the model and structural basis for its cytokine activity. it is predicted that the c-terminus of the full-length protein is tethered to the n-terminal region, thus preventing elr substrate recognition. recombinant human tyrrs is a full-length human tyrosyl-trna synthetase with a mutation at y341a that blocks the h-bond between the elr motif and y as a result, the elr motif is exposed, and mini-rhtyrrs-like activity can then promote thrombocytopoiesis. as a potential thrombocytopoietic drug, large-scale expression and purification of rhtyrrs would be desirable. preclinical testing involving in vitro and animal studies could then be carried out to evaluate its toxic and pharmacologic effects. in this study, rhtyrrs was expressed at a high level in e. coli and purified for future preclinical testing. methods cells and antibodies the competent e. coli strain bl gal dcm; providded by atyr pharma) was used as the host for rhtyrrs expression. this strain was transformed with the pet24a inducible expression vector in which the his-tag sequence was deleted and the t promoter was replaced with a tac promoter. a mouse anti-human il- monoclonal antibody, rabbit anti-human il- polyclonal antibody, and goat anti-rabbit igg-hrp sc- antibody were used. growth conditions a clone with a high rhtyrrs expression level was cultivated in lb agar and m9ca medium. trace element solution consisted of  g feso4 · 7h2o, 2 g mncl2 · 4h2o,  g coso4 · 7h2o,  g cacl2 · 2h2o,  g cucl2 · 2h2o, and  g znso4 · 7h2o. feeding solutions were 50% glycerol, 250 g/l glucose, and 100 g/l yeast extract. the glucose and mgso solutions were sterilized separately. kanamycin sulfate was added to a final concentration of 100 μg/ml in both the m9ca and feeding solutions. fermentation was performed in a 5-l bioflo fermenter with automated control of: the ph at by the addition of ammonium hydroxide, dissolved o at 70% by providing pure oxygen, and agitation at 700 rpm. air was provided at a flow rate of  l/min and the temperature was controlled at 30°c. fermentation was conducted according to the process developed by shiloach et al.. the batch phase ends when cells have used up the available glucose. the best indications that the batch phase has ended include a sharp decrease in stirrer speed and an increase in po the bacterial concentration was measured off-line by the optical density at 600 nm and induced with  mm isopropylthio-β-galactoside once it reached an od of. after 6 h of  mm iptg induction, the cells were harvested by centrifugation at  × g for 30 min. the cell pellet was stored at -70°c. cell lysis harvested cell pellets were resuspended in volumes of 20 mmol/l hac-naac buffer and subjected to two cycles of microfluidization at 1000 bar. the crude extract was then centrifuged at  × g for 60 min. cation exchange chromatography the clarified supernatant was loaded onto a sp sepharose fast flow column that was pre-equilibrated with 20 mmol/l hac-naac buffer. the bound proteins were eluted with a linear nacl gradient. fractions containing rhtyrrs were pooled and analyzed by sds-page. gel filtration the pooled fractions were loaded onto a sephadex-g column pre-equilibrated with 20 mmol/l phosphate buffer and rhtyrrs was washed with 20 mmol/l pb at 5 ml/min. fractions were collected from the column and analyzed by sds-page. anion exchange chromatography the diluted product solution was loaded onto a 50-ml q sepharose fast flow column pre-equilibrated with 20 mmol/l pb. rhtyrrs was eluted with a linear nacl gradient and identified by sds-page via coomassie blue staining. sds-page and sequencing of the n-terminal amino acids electrophoresis was carried out in 1-mm-thick gels using biorad minigel apparatus. coomassie staining was performed as previously described, except that microwave heating was used at each staining step to reduce the total staining and destaining procedure time to 30 min. the protein concentration was measured using bsa as a standard. the sequence of the n-terminal amino acids of purified rhtyrrs was determined using a protein sequencer. western blotting proteins resolved in a pre-cast bis-tris gel were electrotransferred to a pvdf membrane followed by blocking in 10% bsa solution prepared in tbst. the membrane was then incubated with anti-rhtyrrs monoclonal antibody for 90 min at room temperature. after washing, the membrane was incubated with peroxidase-conjugated goat anti-mouse igg for 60 min at room temperature. all antibody incubations and washing steps were carried out in tbst. the immunoreactive bands were visualized with a western blot kit. reversed-phase hplc reversed-phase hplc was used to study the purity of rhtyrrs. a higgins proto c column was used at a flow rate of 1 ml/min and at a temperature of 40°c. the column was eluted with a linear 30-min gradient from 5% b to 95% b in hplc water; b =  % tfa in acetonitrile) and monitored by absorbance at 215 nm with a total run time of 40 min. mass spectrometry analysis the purified protein was analyzed by mass spectrometry. molecular weight measurements were made by lc-ms with a quadrupole-time-of-flight mass spectrometer at the fudan university institute of biomedical science. aminoacylation assay aminoacylation activity was determined at ambient temperature in 150 mmol/l tris–hcl, 150 mmol/l kcl, 10 mmol/l mgcl 10 mmol/l β-mercaptoethanol, 4 mmol/l atp, and 10 mmol/l tyrosine. human trnatyr was prepared as previously described. before each assay, 100 mmol/l human trnatyr was annealed by heating at 65°c for 5 min and cooled to room temperature. reactions were initiated by the addition of rhtyrrs to the reaction mixture. aliquots were taken at fixed intervals and spotted onto whatman filter discs saturated with 5% trichloroacetic acid and dried. the filters were placed into ice-cold 5% trichloroacetic acid and washed three times with fresh 5% trichloroacetic acid at 4°c and once with 95% ethanol. the level of trna aminoacylation was then quantitated by liquid scintillation counting. biological activity assay rhtyrrs activity was tested using elisa to measure il- secretion by rhtyrrs-treated hl- cells. cells were thawed from vials and cultured according to the american type culture collection instructions, except that rpmi- was supplemented with 10% heat-inactivated fetal bovine serum and used as growth medium. cells were plated in a 96-well plate at a density of 1 ×  cells per 1 ml of medium on the day of the assay. the rhtyrrs was added immediately after cell plating. cells were plated at 108 μl- per well. the protein was diluted in sterile phosphate-buffered saline to a concentration of 10 μmol/l. the 10× protein stock was serially diluted and 12 μl was added to each well such that the final protein concentrations were 500 nmol/l, 250 nmol/l, 125 nmol/l,  nmol/l,  nmol/l, and  nmol/l. pbs was used as a control. cells were incubated at 37°c and 5% co for 24 h. the 96-well plate was then spun for 10 min at room temperature in a swinging bucket centrifuge at  × g. the plate was tilted at a 45° angle and 100 μl of media per well was transferred to a new 96-well plate. the amount of secreted il- protein in the media was then determined by sandwich elisa.  rhtyrrs expression and purification to evaluate the long-term stability of the plasmid/strain combination, bacteria were first grown in shake flasks for generations by repeated dilution before the analysis of rhtyrrs expression. a typical growth curve is shown in figure 1a. sds-page analysis of expressed rhtyrrs is shown in figure 1b. the expression level after generations was comparable to that produced by the cells after five generations. approximately –  g/l of rhtyrrs was produced under these growth conditions. when rhtyrrs was expressed in e. coli strain bl using m9ca medium and fed-batch methodology, approximately 50% of the protein was soluble.after 6 h of iptg induction, cells were harvested by centrifugation and lysed by two cycles of microfluidization. the concentrated media were subjected to cation exchange, gel filtration, and anion exchange chromatography, yielding rhtyrrs of up to 97% purity. rhtyrrs was eluted with  mol/l nacl-pb via cation exchange, then subjected to gel filtration to exchange the buffer to pb; all pooled fractions were then loaded onto a 50 ml q sepharose fast flow column pre-equilibrated with 20 mmol/l pb. rhtyrrs was eluted with less than  mol/l nacl-pb during anion exchange. around  g of purified rhtyrrs was generated from 3 l of culture. the products from the optimized rhtyrrs purification process, consisting of cell lysis followed by cation exchange chromatography, gel filtration, and anion exchange chromatography, are shown in figure  rhtyrrs characterization the identity of rhtyrrs was verified by edman degradation to compare the n-terminal sequence of rhtyrrs to the expected mini-tyrrs sequence. up to amino acids from the n-terminus of rhtyrrs were consistently identical to the reference sequence: met-gly-asp-ala-pro-ser-pro-glu-glu-lys-leu-his-leu-ile-trp. we also sought to confirm the identity of rhtyrrs by resolving the sample in parallel with a reference standard by sds-page followed by western blotting using a polyclonal antibody. reversed-phase hplc a reversed-phase hplc was developed as a purity indication assay. figure  shows the analysis of rhtyrrs purified using chromatography steps. this method will be transferred to the contract manufacturer organization or its approved vendor, as appropriate. the retention time of rhtyrrs in the c column was  min with purity up to 97%. mass spectrometry analysis an accurate mass can be detected by intact mass analysis using lc-ms. peaks corresponding to various protonated rhtyrrs species are marked. mass analysis determined that rhtyrrs has a molecular weight of kd. this is identical to that identified by sds-page. aminoacylation assay to test whether rhtyrrs retained the ability to aminoacylate its cognate trna molecule, we took advantage of the inherent synthetase activity of rhtyrrs to verify proper protein folding and assess biological activity during the expression and purification process. the incorporation of 3h tyrosine into trna is a well-established and reproducible assay. biological activity assay we used a sandwich elisa to test il- released by hl- cells in the presence of rhtyrrs. the standard il- concentrations used for elisa were 1200 ng/ml, 600 ng/ml, 300 ng/ml, 150 ng/ml, 75 ng/ml, and  ng/ml as shown in figure 6a, and r was. figure 6b shows the amounts of il- released by hl- cells incubated in the presence of rhtyrrs at concentrations between  nmol/l and 1000 nmol/l, based on the standard il- shown in figure 6a. discussion and  to facilitate the use of rhtyrrs proteins in regulating thrombopoietic activity and to enable structural characterization of this protein, we established a system for the production of large amounts of rhtyrrs. a purity of approximately 97% was routinely achieved with <2% individual contaminants and little to no aggregation. the final, optimized process consists of cell lysis followed by a sequence of three chromatography steps. the first step captures rhtyrrs while contaminants flow through, the second step exchanges the buffer to pb, and the last step removes the remaining process-related impurities. the final concentration is 1 mg/ml in pbs. preliminary characterization of rhtyrrs was performed using western blotting, hplc, mass spectrometry, and n-terminal sequencing to verify the identity of the protein. the ability of the purified rhtyrrs to aminoacylate its cognate trna molecule was also tested, and analysis of the inherent synthetase activity of rhtyrrs was performed to verify proper protein folding and assess biological activity during the expression and purification processes. kinetic parameters from different preparations of rhtyrrs will be compared with a well-characterized reference standard. purification lots that are within the established specifications will be deemed acceptable for release. this assay is still under development but a representative example of the linearity of product accumulation over time is shown below with a range of rhtyrrs concentrations. the y341a substitution activated full-length tyrrs for stimulation of cell proliferation. similar results were observed in an endothelial cell migration assay. in these experiments, wounds were created in monolayers of human umbilical vein endothelial cells. we predict that rhtyrrs not only has il-8-like cytokine activity, but also can enhance il- production from monocytes. the biological assays of hl- stimulation with rhtyrrs presented here demonstrate that this may be the case. the findings presented here will be important for future preclinical studies. competing interests the authors declare that they have no competing interests. authors’ contributions yl, lz and xz performed the protein expression and purification; yz and zf analyzed the protein activity; yl and wm designed the experiments, analyzed data, and wrote the paper; my supervised the experiments and designed the study. all authors read and approved the final manuscript. authors’ information yongjiang lang and yanling zhang are first author. acknowledgments we thank atyr pharma for providing the plasmid, e. coli strain, and the antibody for rhtyrrs. we really appreciate the advise of dr. xianglei yang and dr. paul schimmel from the scripps research institute. this study was supported by the national natural science foundation of china, and science and technology commission of shanghai municipality. this study was also supported by the state key laboratory of biotherapy/collaborative innovation center for biotherapy, west china hospital, sichuan university.
aminoacyl-trna synthetases catalyze the first step of protein synthesis. soluble protein was purified by a three-step purification method using cation exchange chromatography, gel filtration chromatography, and anion exchange chromatography. recombinant human tyrosyl-trna synthetase is engineered with a single amino acid substitution that unmasks its cytokine activity. recombinant human tyrosyl-trna synthetaseexpression of rhtyrrsprotein purificationbiological activity assay
 aminoacyl-trna synthetases catalyze the first step of protein synthesis. emerging evidence indicates that aarss may have additional functions, playing a role in signal transduction pathways regulating thrombopoiesis and inflammation. recombinant human tyrosyl-trna synthetase is engineered with a single amino acid substitution that unmasks its cytokine activity. an industrial production method that provides high yield as well as high purity, quality, and potency of this protein is required for preclinical research.  we expressed codon-optimized rhtyrrs in escherichia coli under fermentation conditions. soluble protein was purified by a three-step purification method using cation exchange chromatography, gel filtration chromatography, and anion exchange chromatography. we also established a method to test the biological activity of rhtyrrs by measuring aminoacylation and il- release in rhtyrrs-treated hl- cells.  the characterization of purified rhtyrrs indicated that this protein can be used in pharmacodynamic and pharmacokinetic studies. recombinant human tyrosyl-trna synthetaseexpression of rhtyrrsprotein purificationbiological activity assay 