BACKGROUND
escherichia coli cells are an efficient factory to produce foreign proteins because of high-cell density, well-known genetics background and the simplest genetic manipulation  <cit> .
however, high expressions of recombinant proteins often result in misfolding, aggregation, degradation, low activity and formation in inclusion-bodies of products, which greatly hampers associated researches and industrial and medical applications  <cit> .
in order to improve the solubility of recombinant proteins, many strategies have been developed, such as co-expression with chaperones, secretion expression of recombinant proteins and protein fusion techniques  <cit> .
in this study, we reported that combined addition of d-glucose and l-arginine in the luria-bertani  medium to cultivate e. coli cells were efficient to enhance the solubility of recombinant arginine deiminases.
arginine deiminase  hydrolyzes l-arginine to l-citrulline, releasing ammonia.
arginine deiminase, combining with ornithine transcarbamylase and carbamate kinase forms the arginine deiminase pathway  <cit> , which is widely distributed in the microorganisms that are able to utilize arginine as an energy source  <cit> .
by depleting extracellular supply of l-arginine, adi is a potential anti-tumor agent for its efficient inhibition effects on the growth of arginine-auxotrophic tumor cells, especially hepatocellular carcinomas and melanomas  <cit> .
adi is also considered a useful producer of l-citrulline at industrial scale  <cit> .
so, many adi genes from different sources, including mycoplasma arginini <cit> , pseudomonas plecoglossicida <cit> , lactococcus lactis <cit> , streptococcus sanguis <cit>  and giardia intestinalis <cit>  have been cloned and expressed in e. coli cells.
however, the recombinant adi proteins were mainly existed in inclusion body forms, which is a bottleneck in its research and development.
we overexpressed arginine deiminases from different resources in the e. coli cells for industrial conversion l-arginine to l-citrulline.
our recombined adi proteins named radi, were similarly mostly in inclusion bodies.
in this study, we made comprehensive optimizations to improve solubility of the radi proteins and found that the combined addition of l-arginine and d-glucose in luria-bertani  medium distinctly increased the in vivo solubility of radi in e. coli cells.
we studied the enzymatic and conversion characteristics of the radi.
RESULTS
construction and expression of adi gene in e. coli
the arginine deiminase of p. putida had been studied due to its characteristics by purification from its original host  <cit> .
because of its high specific activity  and in-vitro solubility, we selected the enzyme to investigate its potential in industrial conversion from l-arginine to l-citrulline.
we optimized the gene sequence according to the codon frequency table of e. coli bl <dig> using the “codon randomization” strategy  <cit>  and synthesized it.
the artificial adi gene  was constructed into different expression plasmids, including pet-30a, pbad/giiia, pcoldtf and pgex-6p- <dig>  for overexpression in e. coli bl <dig>  after a normal optimization process according to the previous report  <cit> ,  <dig>  mm iptg and  <dig> % l-arabinose incubated at 16°c were used to induce the radi expression in e. coli cells.
these expression plasmids, except pet-30a, exhibited nearly no solubility and enzymatic activity to hydrolyze l-arginine to l-citrulline.
the pet30a-adi produced overexpressed radi proteins.
however, there was only a weak corresponding band  in the soluble fractions , which gave the hydrolytic activity of  <dig>  ±  <dig>  u/ml.
most of the expressed proteins were existed in the form of inactive inclusion bodies .
wen et al.
<cit>  reported that truncation of a non-catalytic region in a  <dig> - <dig> -β-d-glucanase from fibrobacter succinogenes improved enzymatic activity and thermotolerance.
the crystal structures of adi from mycoplasma arginini and pseudomonas aeruginosa showed that the enzymes shared the same catalytic triad  and the cys active site  <cit> .
the catalytic region mainly located in the c-terminal region of adi.
to explore the effects of deleting the n-terminal non-catalytic fragment on solubility and activity of radi, our construction based on pet30a-adi, a plasmid that expressed a truncated adi protein lacking the n-terminal  <dig> amino acids .
unfortunately, instead of improving solubility of the product, the truncation resulted in complete inactivation of the enzyme .
co-expression with chaperone teams
it is well known that chaperone proteins, such as dnak-dnaj-grpe and groes-groel are efficient to assist protein folding.
thus, improve the production of active recombinant proteins  <cit> .
we co-expressed the pet30a-adi with four plasmids encoding different chaperone teams respectively.
while pgro <dig>  and pg-kje <dig>  had a similar effect to improve solubility of the radi proteins, pkje <dig>  and ptf <dig>  didn’t play any significant role to improve the solubility .
it suggested that only the groes-groel chaperone team contributed to the improvement of soluble radi proteins.
for example, when the pet30a-adi was co-expressed with pgro <dig>  more radi proteins formed as compared to the single pet30a-adi plasmid that were present in the soluble part .
the total activity was accordingly improved to  <dig>  ±  <dig>  u/ml, approximately 5-fold higher than that of the radi expressed alone.
however, the radi proteins were still mostly in inclusion body forms .
the further optimizations were performed based on the co-expression with pgro <dig>
cultivation-improved solubilization of radi
kozai et al.
<cit>  reported that the addition of l-arginine in the refolding solution was able to clearly increase the enzymatic activity of denatured adi proteins.
however, our experiments showed that the presence of l-arginine had no effect on the in-vitro solubilization of radi proteins in the inclusion body form.
we further tested whether l-arginine is helpful to improve the in vivo solubilization of adi proteins in e. coli cells before the formation of inclusion bodies.
e. coli cells are routinely cultivated in the lb medium, which contains peptones and yeast extracts as the carbon and nitrogen sources.
we added different concentrations of l-arginine in the lb medium to cultivate the pet30a-adi plasmid-harboring e. coli bl <dig> cells.
low concentrations of l-arginine  had no effect on the solubilization of radi proteins, while higher concentrations  caused alkalization of the medium , in which e. coli cells developed poorly or no growth at all.
if we adjusted the media containing high l-arginine concentrations to ph  <dig>  or added l-arginine hydrochloride to replace l-arginine, which did not change the ph of the medium significantly, e. coli cells were able to grow normally but the solubilization of radi proteins did not improve.
the bacteria, such as streptococcus lactis and oenococcus oeni, that are able to utilize l-arginine as energy sources were unable to take up l-arginine in the absence of fermentable sugars  <cit> .
we suggested that less amount of fermentable sugars in the lb medium might cause no effect of the addition of l-arginine on the solubilization of radi proteins.
thus, we supplemented d-glucose together with l-arginine in the lb medium.
interestingly, the expression of soluble adi proteins varied significantly with different concentrations of the supplements.
the combination of  <dig> % l-arginine and  <dig> % d-glucose gave the highest solubility of the protein , the enzymatic activity of the crude extracts reached  <dig>  ±  <dig>  to  <dig>  ±  <dig>  u/ml, which were much higher than that in the other combinations .
under this condition, the yield of total and soluble radi proteins were measured with the bio-rad’ image lab software, increased approximately  <dig>  and  <dig> -fold higher than that of the alone-expressed radi and  <dig>  and  <dig> -fold higher than that of the radi co-expressed with pgro <dig> respectively.
the total activity increased approximately 15-fold higher than the radi expressed alone  and 3-fold higher than that of the co-expression with pgro <dig> .
in the absence of pgro <dig>  the combination of  <dig> % l-arginine and  <dig> % d-glucose also enhanced the total activity to  <dig>  ±  <dig>  u/ml.
combined addition
a
b
a: with the presence of pgro7; b: produced from crude extracts.
it was noted that higher concentrations of d-glucose  supported better growth of cells, but the activities of crude extracts were prominently decreased.
higher concentrations of supplemented l-arginine  inhibited the growth of cells because of alkalization of the medium, even with the presence of d-glucose, which caused no effect in the improvement of enzymatic activities .
for example, at the concentration of 1% l-arginine , the od <dig> of culture broth was less than  <dig>  after 24 hrs of incubation and at the 2% concentration, there were no growth.
surprisingly, when we added different concentrations of l-arginine hydrochloride, instead of l-arginine in the lb medium with d-glucose, no improvement was observed for the solubilization of radi proteins .
when l-arginine was combined with other sugars, such as d-galactose, d-sucrose and d-maltose, the solubilization of radi proteins was not improved.
when l-arginine was replaced with non-substrate amino acids of adi, such as l-tryptophan, l-citrulline and l-glycine no improvement was observed for the solubilization of radi proteins .
we also assayed the effects of the combined addition of  <dig> % l-arginine and  <dig> % d-glucose on the expression of another adi, which was from listeria welshimeri serovar 6b str.
slcc <dig> .
the total activity of the enzyme was  <dig>  ±  <dig>  u/ml in the presence of  <dig> % l-arginine and  <dig> % d-glucose, which was  <dig> -fold higher than it expressed alone  and  <dig> -fold higher than it co-expressed with pgro <dig> .
the increase rate was similar to that for the radi from pseudomonas putida.
these results suggested that the combined addition of l-arginine and d-glucose was applicable in solubilization of different adi proteins.
enzymatic and conversion characteristics of radi
after purification using ni-nta affinity chromatography, the recombinant proteins showed as a single band with a size of 53 kda on sds-page gel .
we assayed enzymatic characteristics of the purified radi proteins.
the radi, which contained some additional terminal fragments such as a his-tag and some restriction sites, showed similar characteristics as the original adi obtained from pseudomonas putida <cit> .
for example, the stability of proteins was observed between ph  <dig>  and ph  <dig>  after 12 hrs incubation .
the optimal ph was  <dig>  for enzymatic activity, but the activity was sharply decreased with the variation of ph value  .
the enzymatic activity of radi was mostly retained after incubation at 30°c or 40°c for 90 min, clearly decreased after incubation at 50°c for 90 min .
cu2+ , sds  and dtt  strongly inhibited the activity of radi, cr3+ , co2+  had a slight inhibition for radi, while other metal ions had no remarkable effect observed on radi .
interestingly, the suitable temperature for the enzymatic activity of radi was between 30°c and 50°c and the optimal temperature was 40°c .
the optimal temperature was 50°c for the original adi measured in pseudomonas putida <cit> .
at the optimal 40°c, the specific activity of radi reached 76 ±  <dig>  u/mg, which was also higher than  <dig>  u/mg of the original at 50°c.we performed conversion experiments using the sodium phosphate buffer  containing 10% l-arginine.
the l-citrulline products increased linearly at 30°c or 40°c.
after 22 hrs of incubation, 20 g of l-arginine substrates were almost converted into l-citrulline by the crude enzyme from 25 ml cultures.
the addition of the batch substrate or not, had no impact on the conversion .
the above enzymatic characteristics suggested that radi was a potential candidate for industrial conversion of l-arginine to l-citrulline.
discussion
previously, it had been observed that the improvement of the substrate l-arginine for correct refolding of denatured adi proteins in the refolding solution  <cit> .
however, l-arginine had no effect on the in vitro solubilization of radi proteins in inclusion body forms.
thus, in vivo solubilization is important for potential industrial applications of radi proteins.
in the laboratories, the e. coli cells are routinely cultivated in lb medium with tryptone and yeast extracts as the carbon and nitrogen sources.
the present study showed that the combined addition of l-arginine and d-glucose in the lb medium significantly improved in vivo solubilization of radi proteins, but the addition of l-arginine or d-glucose alone did not have the effect.
l-arginine have to enter e. coli cells to play the in vivo solubilization functions.
we suggested that the functions of l-arginine in solubilization of adi were probably via the binding to the proteins, thus preventing the formation of inclusion bodies.
the role of d-glucose is still unknown during this process.
there are three possibilities of its roles:  d-glucose helped l-arginine to transport through the cell membrane  d-glucose balanced the medium ph  d-glucose maintained the intracellular ph or metabolic processes.
high concentrations of l-arginine caused alkalization of medium, which extensively inhibited the growth of cells.
however, l-arginine hydrochloride did not change the medium ph significantly.
the addition of the compound had no effect on the solubilization of the radi proteins, even at low concentrations along with d-glucose.
thus, balancing medium ph or intracellular ph is not the function of d-glucose in the in vivo solubilization of radi proteins.
streptococcus lactis and oenococcus oeni are able to utilize l-arginine as a source of energy via the l-arginine catabolism pathway  <cit> .
poolman et al.
<cit>  reported that l-arginine entered these cells by an arginine-ornithine antiporter in s. lactis, which was initiated in the presence of a fermentable sugar.
only after initiated, l-arginine was able to subsequently enter cells via the ornithine and arginine concentration gradients formed during arginine metabolism  <cit> .
it was suggested fermentable sugars provided the energy for the entrance of l-arginine into cells.
bioinformatics analysis showed that the genome of e. coli bl <dig>  had some predicted proteins related to the arginine-ornithine antiporter, such as an arginine/ornithine antiporter transporter, an arginine/ornithine transporter subunit and an arginine/ornithine transport system atpase  <cit> .
we supposed that the increased solubilization was associated with the in vivo interactions between l-arginine and newly synthesized radi proteins during the process of refolding, which requires further determination.
CONCLUSIONS
combined addition of l-arginine and d-glucose in the lb medium significantly improved in vivo solubility of radi proteins but the addition of l-arginine or d-glucose alone did not have the effect.
with the support of groel-groes, the promotion of combined d-glucose and l-arginine was more significant.
the studies present in this paper suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in e. coli cells.

 escherichia coli cells are an efficient factory to produce foreign proteins because of high-cell density, well-known genetics background and the simplest genetic manipulation. however, high expressions of recombinant proteins often result in misfolding, aggregation, degradation, low activity and formation in inclusion-bodies of products, which greatly hampers associated researches and industrial and medical applications. in order to improve the solubility of recombinant proteins, many strategies have been developed, such as co-expression with chaperones, secretion expression of recombinant proteins and protein fusion techniques. in this study, we reported that combined addition of d-glucose and l-arginine in the luria-bertani medium to cultivate e. coli cells were efficient to enhance the solubility of recombinant arginine deiminases. arginine deiminase hydrolyzes l-arginine to l-citrulline, releasing ammonia. arginine deiminase, combining with ornithine transcarbamylase and carbamate kinase forms the arginine deiminase pathway, which is widely distributed in the microorganisms that are able to utilize arginine as an energy source. by depleting extracellular supply of l-arginine, adi is a potential anti-tumor agent for its efficient inhibition effects on the growth of arginine-auxotrophic tumor cells, especially hepatocellular carcinomas and melanomas. adi is also considered a useful producer of l-citrulline at industrial scale. so, many adi genes from different sources, including mycoplasma arginini, pseudomonas plecoglossicida, lactococcus lactis, streptococcus sanguis and giardia intestinalis have been cloned and expressed in e. coli cells. however, the recombinant adi proteins were mainly existed in inclusion body forms, which is a bottleneck in its research and development. we overexpressed arginine deiminases from different resources in the e. coli cells for industrial conversion l-arginine to l-citrulline. our recombined adi proteins named radi, were similarly mostly in inclusion bodies. in this study, we made comprehensive optimizations to improve solubility of the radi proteins and found that the combined addition of l-arginine and d-glucose in luria-bertani medium distinctly increased the in vivo solubility of radi in e. coli cells. we studied the enzymatic and conversion characteristics of the radi.  construction and expression of adi gene in e. coli the arginine deiminase of p. putida had been studied due to its characteristics by purification from its original host. because of its high specific activity and in-vitro solubility, we selected the enzyme to investigate its potential in industrial conversion from l-arginine to l-citrulline. we optimized the gene sequence according to the codon frequency table of e. coli bl using the “codon randomization” strategy and synthesized it. the artificial adi gene was constructed into different expression plasmids, including pet-30a, pbad/giiia, pcoldtf and pgex-6p- for overexpression in e. coli bl after a normal optimization process according to the previous report,  mm iptg and % l-arabinose incubated at 16°c were used to induce the radi expression in e. coli cells. these expression plasmids, except pet-30a, exhibited nearly no solubility and enzymatic activity to hydrolyze l-arginine to l-citrulline. the pet30a-adi produced overexpressed radi proteins. however, there was only a weak corresponding band in the soluble fractions, which gave the hydrolytic activity of  ±  u/ml. most of the expressed proteins were existed in the form of inactive inclusion bodies. wen et al. reported that truncation of a non-catalytic region in a - -β-d-glucanase from fibrobacter succinogenes improved enzymatic activity and thermotolerance. the crystal structures of adi from mycoplasma arginini and pseudomonas aeruginosa showed that the enzymes shared the same catalytic triad and the cys active site. the catalytic region mainly located in the c-terminal region of adi. to explore the effects of deleting the n-terminal non-catalytic fragment on solubility and activity of radi, our construction based on pet30a-adi, a plasmid that expressed a truncated adi protein lacking the n-terminal amino acids. unfortunately, instead of improving solubility of the product, the truncation resulted in complete inactivation of the enzyme. co-expression with chaperone teams it is well known that chaperone proteins, such as dnak-dnaj-grpe and groes-groel are efficient to assist protein folding. thus, improve the production of active recombinant proteins. we co-expressed the pet30a-adi with four plasmids encoding different chaperone teams respectively. while pgro and pg-kje had a similar effect to improve solubility of the radi proteins, pkje and ptf didn’t play any significant role to improve the solubility. it suggested that only the groes-groel chaperone team contributed to the improvement of soluble radi proteins. for example, when the pet30a-adi was co-expressed with pgro more radi proteins formed as compared to the single pet30a-adi plasmid that were present in the soluble part. the total activity was accordingly improved to  ±  u/ml, approximately 5-fold higher than that of the radi expressed alone. however, the radi proteins were still mostly in inclusion body forms. the further optimizations were performed based on the co-expression with pgro cultivation-improved solubilization of radi kozai et al. reported that the addition of l-arginine in the refolding solution was able to clearly increase the enzymatic activity of denatured adi proteins. however, our experiments showed that the presence of l-arginine had no effect on the in-vitro solubilization of radi proteins in the inclusion body form. we further tested whether l-arginine is helpful to improve the in vivo solubilization of adi proteins in e. coli cells before the formation of inclusion bodies. e. coli cells are routinely cultivated in the lb medium, which contains peptones and yeast extracts as the carbon and nitrogen sources. we added different concentrations of l-arginine in the lb medium to cultivate the pet30a-adi plasmid-harboring e. coli bl cells. low concentrations of l-arginine had no effect on the solubilization of radi proteins, while higher concentrations caused alkalization of the medium, in which e. coli cells developed poorly or no growth at all. if we adjusted the media containing high l-arginine concentrations to ph  or added l-arginine hydrochloride to replace l-arginine, which did not change the ph of the medium significantly, e. coli cells were able to grow normally but the solubilization of radi proteins did not improve. the bacteria, such as streptococcus lactis and oenococcus oeni, that are able to utilize l-arginine as energy sources were unable to take up l-arginine in the absence of fermentable sugars. we suggested that less amount of fermentable sugars in the lb medium might cause no effect of the addition of l-arginine on the solubilization of radi proteins. thus, we supplemented d-glucose together with l-arginine in the lb medium. interestingly, the expression of soluble adi proteins varied significantly with different concentrations of the supplements. the combination of % l-arginine and % d-glucose gave the highest solubility of the protein, the enzymatic activity of the crude extracts reached  ±  to  ±  u/ml, which were much higher than that in the other combinations. under this condition, the yield of total and soluble radi proteins were measured with the bio-rad’ image lab software, increased approximately and -fold higher than that of the alone-expressed radi and and -fold higher than that of the radi co-expressed with pgro respectively. the total activity increased approximately 15-fold higher than the radi expressed alone and 3-fold higher than that of the co-expression with pgro. in the absence of pgro the combination of % l-arginine and % d-glucose also enhanced the total activity to  ±  u/ml. combined addition a b a: with the presence of pgro7; b: produced from crude extracts. it was noted that higher concentrations of d-glucose supported better growth of cells, but the activities of crude extracts were prominently decreased. higher concentrations of supplemented l-arginine inhibited the growth of cells because of alkalization of the medium, even with the presence of d-glucose, which caused no effect in the improvement of enzymatic activities. for example, at the concentration of 1% l-arginine, the od of culture broth was less than after 24 hrs of incubation and at the 2% concentration, there were no growth. surprisingly, when we added different concentrations of l-arginine hydrochloride, instead of l-arginine in the lb medium with d-glucose, no improvement was observed for the solubilization of radi proteins. when l-arginine was combined with other sugars, such as d-galactose, d-sucrose and d-maltose, the solubilization of radi proteins was not improved. when l-arginine was replaced with non-substrate amino acids of adi, such as l-tryptophan, l-citrulline and l-glycine no improvement was observed for the solubilization of radi proteins. we also assayed the effects of the combined addition of % l-arginine and % d-glucose on the expression of another adi, which was from listeria welshimeri serovar 6b str. slcc. the total activity of the enzyme was  ±  u/ml in the presence of % l-arginine and % d-glucose, which was -fold higher than it expressed alone and -fold higher than it co-expressed with pgro. the increase rate was similar to that for the radi from pseudomonas putida. these results suggested that the combined addition of l-arginine and d-glucose was applicable in solubilization of different adi proteins. enzymatic and conversion characteristics of radi after purification using ni-nta affinity chromatography, the recombinant proteins showed as a single band with a size of 53 kda on sds-page gel. we assayed enzymatic characteristics of the purified radi proteins. the radi, which contained some additional terminal fragments such as a his-tag and some restriction sites, showed similar characteristics as the original adi obtained from pseudomonas putida. for example, the stability of proteins was observed between ph  and ph  after 12 hrs incubation. the optimal ph was for enzymatic activity, but the activity was sharply decreased with the variation of ph value. the enzymatic activity of radi was mostly retained after incubation at 30°c or 40°c for 90 min, clearly decreased after incubation at 50°c for 90 min. cu2+, sds and dtt strongly inhibited the activity of radi, cr3+, co2+ had a slight inhibition for radi, while other metal ions had no remarkable effect observed on radi. interestingly, the suitable temperature for the enzymatic activity of radi was between 30°c and 50°c and the optimal temperature was 40°c. the optimal temperature was 50°c for the original adi measured in pseudomonas putida. at the optimal 40°c, the specific activity of radi reached 76 ±  u/mg, which was also higher than u/mg of the original at 50°c.we performed conversion experiments using the sodium phosphate buffer containing 10% l-arginine. the l-citrulline products increased linearly at 30°c or 40°c. after 22 hrs of incubation, 20 g of l-arginine substrates were almost converted into l-citrulline by the crude enzyme from 25 ml cultures. the addition of the batch substrate or not, had no impact on the conversion. the above enzymatic characteristics suggested that radi was a potential candidate for industrial conversion of l-arginine to l-citrulline. discussion previously, it had been observed that the improvement of the substrate l-arginine for correct refolding of denatured adi proteins in the refolding solution. however, l-arginine had no effect on the in vitro solubilization of radi proteins in inclusion body forms. thus, in vivo solubilization is important for potential industrial applications of radi proteins. in the laboratories, the e. coli cells are routinely cultivated in lb medium with tryptone and yeast extracts as the carbon and nitrogen sources. the present study showed that the combined addition of l-arginine and d-glucose in the lb medium significantly improved in vivo solubilization of radi proteins, but the addition of l-arginine or d-glucose alone did not have the effect. l-arginine have to enter e. coli cells to play the in vivo solubilization functions. we suggested that the functions of l-arginine in solubilization of adi were probably via the binding to the proteins, thus preventing the formation of inclusion bodies. the role of d-glucose is still unknown during this process. there are three possibilities of its roles: d-glucose helped l-arginine to transport through the cell membrane d-glucose balanced the medium ph d-glucose maintained the intracellular ph or metabolic processes. high concentrations of l-arginine caused alkalization of medium, which extensively inhibited the growth of cells. however, l-arginine hydrochloride did not change the medium ph significantly. the addition of the compound had no effect on the solubilization of the radi proteins, even at low concentrations along with d-glucose. thus, balancing medium ph or intracellular ph is not the function of d-glucose in the in vivo solubilization of radi proteins. streptococcus lactis and oenococcus oeni are able to utilize l-arginine as a source of energy via the l-arginine catabolism pathway. poolman et al. reported that l-arginine entered these cells by an arginine-ornithine antiporter in s. lactis, which was initiated in the presence of a fermentable sugar. only after initiated, l-arginine was able to subsequently enter cells via the ornithine and arginine concentration gradients formed during arginine metabolism. it was suggested fermentable sugars provided the energy for the entrance of l-arginine into cells. bioinformatics analysis showed that the genome of e. coli bl had some predicted proteins related to the arginine-ornithine antiporter, such as an arginine/ornithine antiporter transporter, an arginine/ornithine transporter subunit and an arginine/ornithine transport system atpase. we supposed that the increased solubilization was associated with the in vivo interactions between l-arginine and newly synthesized radi proteins during the process of refolding, which requires further determination.  combined addition of l-arginine and d-glucose in the lb medium significantly improved in vivo solubility of radi proteins but the addition of l-arginine or d-glucose alone did not have the effect. with the support of groel-groes, the promotion of combined d-glucose and l-arginine was more significant. the studies present in this paper suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in e. coli cells. 
overexpression of foreign genes in escherichia coli cells is an efficient means to obtain recombinant proteins. the present study suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in e. coli cells. after codon optimization, we synthesized the adi gene of pseudomonas putida and constructed it for overexpression in e. coli cells. the enzymatic and conversion characteristics of the radi products were further determined. combined addition of l-arginine and d-glucose in the lb medium significantly improved in vivo solubility of radi proteins. co-expression with the groes-groel chaperone team increased approximately 5-fold of the radi activity. the radi products were mainly in inclusion body forms. the technique is, however, often hampered by misfolding, degradation, aggregation and formation in inclusion bodies of products. in this study, we reported that in vivo solubility of overexpressed arginine deiminases improved by changing the cultivation conditions. in addition the combination of l-arginine and d-glucose in the luria-bertani growth medium further increased the total activity to about times.
 overexpression of foreign genes in escherichia coli cells is an efficient means to obtain recombinant proteins. the technique is, however, often hampered by misfolding, degradation, aggregation and formation in inclusion bodies of products.  in this study, we reported that in vivo solubility of overexpressed arginine deiminases improved by changing the cultivation conditions. adi is enzymes that convert l-arginine to l-citrulline. after codon optimization, we synthesized the adi gene of pseudomonas putida and constructed it for overexpression in e. coli cells. the radi products were mainly in inclusion body forms. we performed a series of optimization to enhance solubility of the protein. co-expression with the groes-groel chaperone team increased approximately 5-fold of the radi activity. in addition the combination of l-arginine and d-glucose in the luria-bertani growth medium further increased the total activity to about times. separate l-arginine and d-glucose or the addition of other saccharides or amino acids had no such effects. the solubilization effects of the combination of l-arginine and d-glucose were further confirmed in the overexpression of another adi from listeria welshimeri. the enzymatic and conversion characteristics of the radi products were further determined.  combined addition of l-arginine and d-glucose in the lb medium significantly improved in vivo solubility of radi proteins. the present study suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in e. coli cells. arginine deiminaseheterologous expressionescherichia colisolubilizationcultivationconversionl-argininel-citrulline 