thymosin alpha 1 ( t1 ) , a 28-amino - acid peptide with molecular weight ( mw ) of 3108 da , 
 was first isolated from calf thymus in 1977 by goldstein et al . [ 1 , 2 ] .
t1 is 
 mainly produced in vivo by thymic epithelial cells and is stable 
 at 8090c .
t1 has no aromatic amino acids ( see 
 figure 1 ) , and n - acetylation is important for protecting towards proteolysis .
t1 
 plays a role against viral infection , against tumors , and in 
 immunomodulation . upon stimulation of t1 ,
t cells 
 become mature in vitro and express il-2 , il-2r , and cd2 [ 4 , 5 ] .
t1 inhibits viral replication at 
 least in hepatitis virus b - transfected hepg2 tumor cells [ 6 , 7 ] .
in addition , t1 
 antagonizes t cell apoptosis induced by dexamethasone , cd3 monoclonal antibody treatment 
 , or addition of sera from 
 tumor - bearing mice
. it also downregulates tumor weight 
 in mice , enhances major 
 histocompatibility complex ( mhc ) class i antigen expression in tumor 
 cells and antigen - presenting cells [ 1113 ] , and partly restores cellular 
 immunity .
t1 is used in combination with 
 ifn- for patients with chronic hepatitis virus b or c infection [ 15 , 16 ] .
recently , t1 has even been 
 used as an adjuvant for vaccines against influenza and hepatitis b . in the past two decades , although the successful expression of the recombinant t1 ( r t1 ) and prothymosin ( thymosin comprises the n - terminus of prothymosin ) in escherichia coli has 
 been reported [ 19 , 20 ] .
however , t1 was 
 expressed with a fusion protein or in a concatemer form [ 21 , 22 ] .
the expression of the 
 t1 monomer peptide has not been reported , because it is difficult to extract the micromolecular 
 peptide from the fermentation broth . the fusion protein
all of these issues made it difficult 
 to prepare active recombinant t1 . in this study , we have expressed t1 concatemer 
 protein and cleaved the concatemer protein with hydroxylamine to obtain active t1 
 monomer peptide .
the pet-22b ( + ) vector , e. coli bl21 
 ( de3 ) ( hsds gal [  
 cits857 ind1 sam7 nin5 lacuv5-t7 ] ) , the hepg2 liver carcinoma cell line , and the spc - a-1 
 lung carcinoma cell line were kindly provided by institute of biochemistry and 
 cell biology ( shanghai , cas , china ) .
hepg2 and spc - a-1 
 cells were cultured in rmpi 1640 medium with 10% fetal bovine serum in 
 humidified air at 37c with 5% 
 co2 .
lymphocytes were isolated from adult mouse spleens ( bal / c ) and 
 cultured in rmpi 1640 medium with 10% fetal bovine serum in humidified air at 37c with 5% co2 . synthesized t1 
 ( sinopep pharmaceutical , inc . , hangzhou , china . )
the synthesized t1 and the 
 cleaved t1 were dissolved in phosphate - buffered solution 
 ( pbs ph 7.2 ) at 1 mg / ml ( approximately 3  10
two single strand dna oligomers encoding the entire 
 thymosin alpha peptide ( synthesized by songon , shanghai , china . ) , partially complementary to 
 each other ( as indicated by the underlined sequences ) were synthesized .
thy - f : gtcgacatgggatccaacggttctgatgctgctgtagatacttcttctgagattactactaaagacctaa 
  thy - r : ctcgagtcaagatctcccgttctcagcctcttcgacaacttccttcttctcctttaggtctttagtagta 
  the 2 oligonucleotides were mixed and denatured by incubation at 94c   for 10 minutes , 
 and subsequently annealed at 60c for 10 minutes in presence of taqplus ( bbi , toronto , canada ) . 

the desired double - strand dna was treated with t4 polynucleotide kinase ( bbi , toronto , 
 canada ) and cloned into the cloning vector pucm - t ( bbi , toronto , canada ) .
the plasmid ( pucm - t - t1 , pt - t1 ) containing the t1 gene was digested with bamh i / xho i or bgl ii / xho i , respectively .
the digested fragments containing 
 the coding sequence of t1 were ligated with t4 dna ligase for 16 hours at 4c to produce the concatemer t1 gene of 2 repeats ( pt - t12 ) . when 
 the plasmid was digested with isocaudamer bamhi ( gctag c ) and bgl ii ( agatc 
 t ) , the 2 fragments had similar termini .
when the 2 fragments were catalyzed 
 with t4 dna ligase the new sequence formed was ggatct , and this new site could 
 not be digested by bamh i or bgl ii ( see figure 2 ) . using a pt - t12 plasmid , plasmids containing concatemer
4 
 repeats ( pt - t14 ) of the t1 gene were 
 prepared as described above . using a pt - t12 plasmid and a pt - t14 plasmid , 
 the preparation of plasmids containing concatemer 6 repeats ( pt - t1  6 ) was the 
 same as pt - t12 plasmid . finally , a fragment containing the concatemer t1 gene of 6 repeats was constructed and 
 inserted into expression vector pet-22b ( + ) , 
 between the restriction endonuclease recognition sites hind iii 
 and nde i. a single bacterial colony containing pet-22b ( + ) -t1
was grown 
 overnight at 37c in 5 ml 
 lb medium with 50 g / ml ampicillin ( bbi , toronto , canada ) .
the overnight culture was 
 inoculated into 250 ml lb medium in a 1 l flask with 50 g / ml ampicillin . 

after 2 hours of incubation , isopropyl-1-thio - beta - d - galactoside ( iptg ; bbi , canada ) 
 was added to the culture at a final concentration of 1.0 mm , when the cell density had 
 reached od600 nm = 0.6 .
after incubation at 37c for 8 hours , the bacterial cells were harvested by centrifugation ( 5000  g , 15 minutes ) at 4c .
the inducibility of the concatemer gene 
 was determined by sodium dodecylsulfate - polyacrylamide gel electrophoresis 
 ( sds - page ) ( data not shown ) .
the cell pellet was resuspended in 100 ml buffer a ( 50 mm tris - hcl [ ph 8.0 ] , 1 mm edta [ ph 8.0 ] , and 0.1 m nacl ) and then sonicated ( 800 w , 20 minutes ) . 

after sonication , the sedimented material ( containing the concatemer protein ) was 
 washed once first with 100 ml buffer b ( 50 mm tris - hcl [ ph 8.0 ] , 1 mm edta ( ph 8.0 ) , and 0.5% triton - x 100 ) and then with 100 ml buffer 
 c ( 50 mm tris - hcl [ ph 8.0 ] , 1 mm edta ( ph 8.0 ) , and 2 m urea ) by centrifugation . the sedimented 
 material
was then dissolved in 100 ml buffer d ( 50 mm tris - hcl [ ph 8.0 ] , 1 mm edta [ ph 8.0 ] , and 8 m urea ) with stirring for 1 hour at 25c .
the 
 soluble protein was then dialyzed ( dialysis membrane mwco 10 000 , spectrum , tex , usa ) with double distilled h2o and cryodesiccated . the concatemer protein
the cleavage buffer   contained 0.1 m trizma base and 2.0 m hydroxylamine - hcl .
the ph of the 
 cleavage buffer was adjusted with 5 m naoh solution to a ph of 9.0 , and the reaction was performed at 45c for 4 hours .
the reaction was terminated 
 by adjusting the ph to 4.0 with concentrated formic acid and cooling to 4c .
using sephadex - g25 ( ge ) column 
 chromatography , the 
 products of cleavage were desalted .
column chromatography revealed 2 
 peaks ; one peak corresponded to the t1 monomer and the other contained the incompletely 
 cleaved concatemer protein .
the molecular mass of the t1 monomer was analyzed 
 by gly - sds - page and mass spectrometry .
spleens were removed from mice and dispersed through 
 nylon mesh to generate a single - cell suspension .
cells were pelleted ( 1500  g , 6 minutes ) , and the cell pellet was 
 resuspended in erythrocyte lysis solution ( 0.15 m nh4cl , 0.02 m tris , sterilized with a 0.22 m filter ) for 5 minutes to remove the red cells . 
 after lysis , the cell suspension was incubated in a culture flask for 2 hours , 
 and then the suspension was transferred to a new flask .
splenic lymphocytes were incubated in rpmi 
 1640 medium with 5 g / ml concanavalin a ( cona ; bbi , canada ) for 12 hours , and 
 then harvested by centrifugation ( 1500  g , 6 minutes ) .
the cell pellet 
 was resuspended in rpmi 1640 medium at a concentration of 1  10 cells / ml . a 96-well 
 culture plate ( costar , inc .
, 
 calif , usa ) was used for incubation of 
 180 l lymphocytes in rpmi 1640 medium .
50 g / ml of the proteins 
 ( cleaved t1 and 
 synthesized t1 ) were added to the wells .
after 24-hour or 48-hour incubation 
 at 37c , 20 l 
 of mtt ( 0.5 mg / ml ) , solution was added to each well , and the microplates were 
 incubated for 4 hours at 37c . 

after incubation , the plates were centrifuged ( 1400  g , 18c , 10 minutes ) .
the supernatants were 
 removed , and 200 l of dimethyl sulfoxide ( dmso ) was added to 
 each well and incubated for 15 minutes at room temperature .
after 
 incubation , the solubilized reduced mtt was measured colorimetrically at 
 570 nm using a benchmark microculture plate reader ( bio - rad , calif , usa ) .
the influence of t1 on mitochondrial activity was measured using mtt reduction , a 
 common experimental method to quantify mitochondrial activity .
the cells were 
 seeded in a 96-well culture plate at 1  10 cells / ml . 
 after 24-hour , 48-hour , and 72-hour exposures to t1 at a concentration of 0.1 mg / ml , 
 the cells were incubated with mtt ( 0.5 mg / ml ) for 4 hours at 37c .
the formazan precipitate 
 was then dissolved in 200 l dmso , and absorbance at 570 nm was measured using the benchmark microculture plate reader ( bio - rad , calif , usa ) . the data are presented as mean  sem , for at least 3 replicate 
 experiments and evaluated using the student 's t - test .
p - values below 0.05 were regarded as statistically 
 significance ( two - tailed test ) .
hydroxylamine - induced cleavage 
 at the asp - gly dipeptide site inserted between the 2 moieties of recombinant 
 fusion proteins had been used for both analysis and preparation of mature 
 protein .
cleavage occurs at the asn - gly bonds and results from 
 the tendency of the asp side chain to cyclize the formation of a substituted 
 succinimide that is susceptible to nucleophilic attack by hydroxylamine .
the 
 infrequency of asn - gly bonds in most proteins results in the production of very 
 large fragments that may overlap cnbr - produced fragments . in the complete amino acid 
 sequence of t1  
( see figure 1 ) , only 1 asp was 
 found at the c - terminus .
hydroxylamine - induced 
 cleavage is high specificity and safe , however , the efficiency of 
 hydroxylamine cleavage is low .
so we expressed a concatemer protein of 6 
 repeats of t1 
 to increase the yield of t1 after cleavage and to decrease the cleavage sites .
the optimal conditions for inducing production of recombinant concatemer protein were 
 at an iptg concentration of 1.0 mm 37c for 8 hours . under these 
 conditions , the recombinant protein ( ~23 kd ) comprised 31% of the total 
 bacterial protein ( see figure 4(a ) ) .
the concatemer protein was identified 
 primarily in the inclusion bodies ( see figure 4(b ) ) .
g-25 column chromatography was used to not only desalt but 
 also remove the incompletely cleaved concatemer protein . because the molecular weight of the t1 
 monomer was ~3.1 kd
however , the incompletely cleaved concatemer 
 proteins , including 2t1 ( ~7 kd ) , 3t1 ( ~11 kd ) , 4t1 ( ~15 kd ) , 
 5t1 ( ~19 kd ) , and 6t1 ( ~23 kd ) , could not enter the g-25 gel because 
 the gel only allows the entry of molecules < 5 kd .
the gly - sds - page could satisfactorily 
 separate polypeptides with molecular weights ranging from 3.5 to 68.0 kd with 
 good linearity , and demonstrated the successful hydroxylamine - induced cleavage 
 of the concatemer proteins ( see figure 5 ) .
however , not all the concatemer proteins were cleaved , and the 
 incompletely cleaved proteins could be completely cleaved by another reaction ( data 
 not shown ) .
ms was performed to analyze the exact 
 molecular weights of the products and the synthesized t1 .
the result showed that 
 the products had only 1 peak of m / z = 1042.19 , while the synthesized t1 had 2 
 peaks of m / z = 1037.80 and 1555.06 ( see figure 6 ) .
the molecular weight of the 
 synthesized t1 was found to be approximately 3108 da , it was assumed that the 
 peptide might attach 2 or 3 protons [ ( 1037.801 )  3 = 3110.4 ; 
 ( 1555.061 )  2 = 3108.12 ] . 

therefore , the cleaved t1 was believed to attach 3 protons , and the molecular 
 weight of the products was approximately 3123.57 [ ( 1042.19 - 1 )  3 = 3123.57 ] .
the 
 difference in the molecular weights between the cleaved t1 and the synthesized 
 t1 was approximately 15 . according to a previous research , the structural difference 
 between the products and
the synthesized t1 was a nh added to the 
 n - terminus 
 of the cleaved peptide ( see figure 9 ) . to determine the bioactivity of the t1 monomer on lymphocyte 
 proliferation ,
the mitochondrial activity was compared in the presence with the 
 absence of the t1 .
preliminary results 
 showed that the peptides could stimulate the mice spleen lymphocyte 
 proliferation .
after a 24-hour treatment with t1 cleaved by hydroxylamine , 
 mitochondrial activity was upgraded to 119.1% and after 48 hours , it was 110.4%.the 
 numbers of the synthesized t1 were 117.5% and 116.0% at 24 hours and 48 hours , 
 respectively ( see figure 7 ) .
both of the peptide preparations could stimulate the 
 proliferation of mice spleen lymphocytes to a similar degree . in this study , we used human liver tumor cells hepg2 
 and human lung tumor cells spc - a-1 as model , because the liver tumor and lung 
 tumor are the most general tumor . to determine the role of mitochondria during incubation , 

the mitochondrial activity was examined . after the treatment with the cleaved t1 
 for 24 hours , 48 hours , and 72 hours , the mitochondrial activity of the spc - a-1 
 cells decreased to 82.4% , 27.1% , 11.6% , respectively , and the corresponding 
 activities with synthesized t1 were 62.0% , 15.1% , and 9.8% , respectively .
on 
 the other hand , after the treatment for 24 hours , 48 hours , and 72 hours , the 
 mitochondrial activity of hepg2 cells decreased to 86.6% , 18.4% , and 10.4% , 
 respectively , and corresponding activities with the synthesized t1 were 59.2% , 
 13.1% , and 10.0% , respectively ( see figure 8) .
both of the peptide preparations 
 decreased the mitochondrial activity remarkably , as compared to the control . 

the difference in the activity between the 2 peptides was not significant ; 
 however , the process of synthesizing t1 was rather intricate .
t1 is mainly obtained by chemical synthesis and animal thymus 
 extraction . however , in fact , t1 from an animal is often impure and 
 may therefore cause heterogeneous allergy . although chemically synthesized t1 can reach a high level of purity , it is necessary to eliminate the side products at each step , including incorrect joining and dextral compounds .
the 
 longer the peptide is , the more intricate the chemical synthesis process becomes .
t1 
 comprises 28 amino acid residues , and its chemical synthesis incurs a high production cost .
prokaryotic 
 expression systems allow for high expression of foreign genes and are widely used for producing large 
 amounts of proteins or peptides . in this research , using a prokaryotic 
 expression system bl21 ( de3)/pet-22b ( + ) , we have established a new method to 
 obtain relatively pure and large quantities of t1 .
when the plasmid was digested with isocaudamer bamh i ( gctag 
 c ) and bgl ii ( agatc t ) , the 2 fragments had similar termini . when the 2 fragments 
 were catalyzed with t4 dna ligase , the new sequence formed was ggatct , and this 
 new site could not be digested by bamh i or bgl ii ( see figure 2 ) .
therefore , the 
 new 2  concatemer gene could not be destroyed when the 4  concatemer gene was 
 constructed .
the new site ggatct corresponded to the dipeptide gly - ser , located at the downstream of the t1 gene .
when concatemer protein was cleaved with hydroxylamine , 
 the downstream amino acid of t1 peptide was removed from this gly site .
when 
 the cleavage reaction proceeded , the upstream amino acid of t1 peptide was 
 removed from another gly site , which was added in front of the t1 peptide .
it is common knowledge that extraction of the micromolecular recombinant peptide 
 from fermentation broth is difficult , when t1 is expressed alone .
generally , a 
 fusion protein is used under these circumstances , for example , a dsba fusion protein .
however , the fusion protein must be 
 removed because the peptide might be inactive , and this process incurs a high production cost .
the concatemer protein of 6 
 repeats6t1was prepared in 
 this research to avoid the use of a fusion protein when expressing the recombinant 
 protein , but the number of the repeats was not unique .
proteins with 4 , 8 , 12 , and 
 so forth repeats could also be constructed .
the longer the concatemer protein 
 is , the greater the yield of the t1 monomer is . on the other hand , when a concatemer protein 
 with more repeats is cleaved , the amount of the incompletely cleaved protein also 
 increases ( see figure 5 ) .
however , these incompletely cleaved proteins can be 
 cleaved by other 1 or 2 rounds of hydroxylamine cleavage .
hydroxylamine 
 was used to cleave the asn - gly peptide bond between the fusion protein and 
 target protein . in this study ,
hydroxylamine was used to cleave the concatemer 
 protein of 6t1 to release the t1 monomer .
while chemical cleavage methods 
 use relatively inexpensive and widely available reagents , cleavage conditions 
 are often relatively harsh and amino acid side - chain modifications may occur . 

this is especially true when hydroxylamine is used to cleave the asn - gly 
 peptide linkages as other asn or gln residues can be modified to their 
 hydroxamic acid forms . because the t1 peptide does not contain gln , met , or other asn , chemical 
 heterogeneity dose not occur by conversion of the gln or asn to their 
 hydroxamate forms .
moreover , there is no possibility of chemical heterogeneity by 
 conversion of met to its oxidized forms .
after hydroxylamine cleavage , a gly remained at the n - terminus of t1 . however , the natural t1 
 had an acetylated nh - terminal ( see figure 9 ) .
the molecular weight 
 difference between the 2 groups was 15 da , but there was no significant difference 
 between the 2 bands revealed by the gly - sds - page ( see figure 5 ) .
hence , we used 
 an ms to analyze the exact molecular weights of the 2 peptides .
the result 
 showed a difference was 15 da ( 31233108 da ) ( see figure 
 6 ) , indicating that the concatemer protein was cleaved satisfactorily . to investigate the influence of the extra 
 imino group on the t1
, we compared the antitumor and immunomodulation activities between 
 the synthesized t1 and the cleaved t1 .
furthermore , we found that t1 
 was able to depress the prolife ration 
 of hepg2 cells by downregulating the oxidative stress ( data not shown ) .
jrnvall 
  had analyzed 40 known 
 -amino - acetylated polypeptide chains and postulated that acetylation occurs 
 during an early posttranslational stage and may have particular importance in 
 this regard .
however , the structural or functional significance of the n - terminal 
 acetylation of t1 is not 
 known , and a protective function of acetylation with regard to proteolysis is 
 possible .
the change in the n - terminus of 
 cleaved peptides might not influence the bioactivity of t1 ; however , it might 
 influence the stability in vivo . in summary , t1 
 with a novel structure was prepared via the expression and hydroxylamine 
 cleavage of a t1 concatemer protein .
this novel t1 could perform proliferation 
 of mice splenic lymphocytes activities , identical to the natural t1 .
this 
 study would provide the basis for the preparation of active t1 and other 
 peptides .
 human thymosin alpha 1 ( t1 ) is an important peptide in the development and senescence of immunological competence in human , and many studies have reported the expression of this peptide . in this study , we designed and synthesized the t1 gene according to the e. coli codon usage preference and constructed a 6t1 concatemer . 
 the latter was inserted into an e. coli expression vector pet-22b ( + ) , and transformed into e. coli bl21 ( de3 ) . 
 after induction with iptg , the concatemer protein was successfully expressed in e. coli then cleaved by hydroxylamine to release the t1 monomer . 
 gly - sds - page and mass spectrometry confirmed that the recombinant protein was cleaved as intended . 
 the bioactivity of the t1 monomer was analyzed by lymphocyte proliferation and by mitochondrial activity in two different tumor cell lines . 
 this study provides a description of the preparation of a bioactive t1 , which may prove useful in future biomedical research . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION