spermatogenesis is a complex process of development that occurs in most mammals throughout adulthood period . this process happens in seminiferous tubules , lined with epithelial tissue that contains sertoli cells . sertoli cells are surrounded by a thin septum of peritubular cells that hold keep germ cells ( 1 , 2 ) . the spermatogonia undergo mitotic divisions , meiotic alternations , and morphologically transform to highly developed cells , spermatozoa , with a unique function and structure ( 3 ) . such a complex process needs a unique program to control and regulate the involved genes . this gene regulation is accomplished by cell surface molecules which participate in cell to cell and cell to extracellular matrix interactions ( 4 , 5 ) . on the other hand , when sperm is passing through the male and female genital tracts , it gains fertilization ability ( capacitation ) and adopts maturity characteristics . maturity is initiated in the epididymis , where sperm communicates with the cell surface molecules of the pathway ( 6 ) . therefore , the investigation of stage and/or cell - specific molecular expression in testis or post - testicular maturation system can improve our understanding of germ cell differentiation and sperm formation ( 7 ) . first identified a 38kda protein ( mtex101 ) in adult mouse testis which is detectable on the surface of spermatocytes and spermatids in testis that it is absent on somatic cells , including sertoli or interstitial cells , like leydig cells ( 3 ) . there is a temporary expression of mtex101 on oogonia , but the molecule disappears in the mature ovary . in embryonic testis , mtex101 is detected on the prosperm - atogonia ( 8) . upon puberty , mtex101 mrna is expressed in spermatocytes and in steps 1 - 9 spermatids during spermatogenesis , but not during spermatogonia . the tex101 protein remains on the cell surfaces in steps 10 - 16 spermatids and testicular sperm , but hides from epididymal sperm after passing the caput epididymis ( 6 ) . the expression of mtex101 homologous protein has been reported in a leukemia cell line and lung cancer in rats and humans , respectively ( 9 , 10 ) . the exact structure and function of mtex101 is not completely understood yet , but it has been speculated that this protein is associated with several transmembrane proteins like ly6k 
 ( 11 ) and cellubrevin ( 12 ) that transduce the extra - cellular signals to the intracellular molecules . the present study was undertaken to produce recombinant mtex101 , which might pave the way for the production of specific antibodies for further structural and functional characterization of the molecule . total rna was isolated from male gonadal organs ( four to 6 week - old mice from pasteur institute of iran ) . rna concentration was measured by a biophotometer ( eppendorf , hamburg , germany ) at 260 nm . one microgram of the total rna was reversetranscribed to cdna by using 200u of molony murine leukemia virus ( rtm - mulv ) reverse transcriptase enzyme ( fermentas , vilnius , lithuania ) , and 20pmol of random hexamer primers ( cybergene , stockholm , sweden ) . pcr amplifications for mtex101 transcript was performed in a volume of 25l using 10 - 20ng of testis cdna , forward and reverse primers ( 10pmol each ) , specific for mtex101 transcript , 10x pcr buffer ( 2.5l ) , dntp mixture ( 0.2 mm each ) , 1 mm mgcl2 , and 1 unit of taq dna polymerase ( roche , mannheim , germany ) . pcr reactions were carried out using a thermal cycler ( eppendorf mastercycler gradient ) as indicated below : a preheating cycle at 95c for 3min , 35 cycles of denaturation at 95c for 30sec , annealing at 60c for 30sec , extension at 72c for 60sec , and finally a 7-min cycle at 72c . the forward and reverse mtex101 orf primers used ( forward : 5ccg aat tca tgg gag cct gcc gca tcc ag 3 , reverse : 5 agg gaa gtg ggt gag ggg gga gca gag cgg ccg c 3. ) , contained ecori and noti restriction enzyme ( re ) sites , respectively . the pcr products were resolved by ethidium bromide - stained 1.5% agarose gel electrophoresis , which revealed a single distinct band at 750bp ( figure 1 ) . the 750bp band was purified using qia quick gel extraction kit ( qiagen , germantown , md , usa ) . pcr optimization on mouse testis cdna by mgcl2 gradients . 1 - 4 : mgcl2 concentrations from 1 to 4 mm , respectively , 5 : 1 kb dna ladder , 6 : negative control ( no dna ) . the pgem - t easy vector ( promega , madison , wi , usa ) was used for the cloning of the purified pcr product . ligation reac tion was set up by 50ng of pgem - t easy vector ( promega ) , three units of t4 dna ligase , 75ng of mtex101 purified fragment , and 6l of rapid 2x ligation buffer ( promega ) . competent cells of e. coli jm109 strain were used for transformation through heat shock method ( 17 ) . the transformed bacteria were left for 1 hr in lb broth at 37c for recovery , and later 100l of the transformation culture was plated onto an ampicillin ( 100mg / ml ) ( sigma , louis , mo , usa ) , iptg ( sigma ) ( 0.5 mm ) and x - gal ( sigma ) , ( 80g / ml ) containing lb agar plate and was cultured for 16hrs at 37c . the recombinant plasmids were isolated from confirmed colonies by miniperp kit ( qiagen ) and then digested by ecori and noti restriction enzymes . plasmid dna ( 800ng ) was used in 25l of the total volume , including noti ( 15units ) , ( invitrogen , carlsbad , ca , usa ) , ecori ( 15 units ) , ( invitrogen ) , and 2.5l of 10 x reaction buffer 3 ( invitrogen ) and incubated for 1.5 hr at 37c . the mtex101 fragment in pgem - t easy was extracted form agarose gel and then subcloned into a pet-28a ( + ) expression vector ( merck , darmstadt , germany ) and was digested by the same restriction enzymes above . ligation reaction was performed by 125ng of digested pet-28a ( + ) , 0.5pmol of mtex101 purified fragments , 2l of rapid 2x ligation buffer ( pro - mega ) , and 3 units of t4 dna ligase ( promega ) in 10l of the total volume and it was incubated overnight at 4c . ligation products were used for transformation of e. coli jm109 strain by heat shock method ( 17 ) . after a recovery time of 1 hour in lb broth , the transformed bacteria were cultured on lb agar and kanamycin ( 50g / ml ) ( sigma , usa ) containing plates for 16 hrs at 37c . the obtained colonies were screened by colony pcr and one of them was confirmed by dna sequencing . recombinant plasmid was purified and was then used for transformation of bl21 ( de3 ) , which is a protease deficient strain of e. coli . a single transformed colony was inoculated into 50ml of lb broth , including kanamycin ( 50g / ml ) ( sigma , usa ) and untransformed bl21 ( de3 ) bacteria were cultured into 50 ml of lb medium . incubation was performed under shaking at 37c until the optical density ( od ) at 600 nm reached 0.6 . iptg ( sigma ) was added to the remainder with the final concentration of 1 mm and incubation continued for 2 - 3 more hours . cell lysates were prepared by sonication of pellets in pbs buffer including 1% protease inhibitor ( roche ) . protein solutions were obtained from bacteria by boiling them in a sample buffer that contained tris - hcl ( ph=6.8 , 0.5 m ) , sds ( 10% w / v ) , glycerol ( 50% v / v ) , and bromo phenol blue ( 0.5% w / v ) , for 5min . samples ( 50l of each ) were separated by sds - page ( 8% ) ( bio - rad , hercules , ca , usa ) under non - reducing conditions . the protein constituents were then electro - phoretically blotted onto a polyvinylidene difluoride ( pvdf ) membrane ( millipore , billerica , ma , usa ) , as described by towbin et al . 
 ( the membrane was blocked in 5% skim milk / 0.1% tween 20 in pbs ( ph 7.4 ) , and then the reactivity of the transferred protein(s ) with 1g / ml of anti mtex101 pab ( avicenna research institute , iran ) was assessed using sheep anti rabbit ig hrp ( avicenna research institute , iran ) . finally , the membranes were visualized using an ecl system ( ge healthcare , biotech , bucking - hamshire , uk ) . total rna was isolated from male gonadal organs ( four to 6 week - old mice from pasteur institute of iran ) . rna concentration was measured by a biophotometer ( eppendorf , hamburg , germany ) at 260 nm . one microgram of the total rna was reversetranscribed to cdna by using 200u of molony murine leukemia virus ( rtm - mulv ) reverse transcriptase enzyme ( fermentas , vilnius , lithuania ) , and 20pmol of random hexamer primers ( cybergene , stockholm , sweden ) . pcr amplifications for mtex101 transcript was performed in a volume of 25l using 10 - 20ng of testis cdna , forward and reverse primers ( 10pmol each ) , specific for mtex101 transcript , 10x pcr buffer ( 2.5l ) , dntp mixture ( 0.2 mm each ) , 1 mm mgcl2 , and 1 unit of taq dna polymerase ( roche , mannheim , germany ) . pcr reactions were carried out using a thermal cycler ( eppendorf mastercycler gradient ) as indicated below : a preheating cycle at 95c for 3min , 35 cycles of denaturation at 95c for 30sec , annealing at 60c for 30sec , extension at 72c for 60sec , and finally a 7-min cycle at 72c . the forward and reverse mtex101 orf primers used ( forward : 5ccg aat tca tgg gag cct gcc gca tcc ag 3 , reverse : 5 agg gaa gtg ggt gag ggg gga gca gag cgg ccg c 3. ) , contained ecori and noti restriction enzyme ( re ) sites , respectively . the pcr products were resolved by ethidium bromide - stained 1.5% agarose gel electrophoresis , which revealed a single distinct band at 750bp ( figure 1 ) . the 750bp band was purified using qia quick gel extraction kit ( qiagen , germantown , md , usa ) . pcr optimization on mouse testis cdna by mgcl2 gradients . 1 - 4 : mgcl2 concentrations from 1 to 4 mm , respectively , 5 : 1 kb dna ladder , 6 : negative control ( no dna ) . the pgem - t easy vector ( promega , madison , wi , usa ) was used for the cloning of the purified pcr product . ligation reac tion was set up by 50ng of pgem - t easy vector ( promega ) , three units of t4 dna ligase , 75ng of mtex101 purified fragment , and 6l of rapid 2x ligation buffer ( promega ) . competent cells of e. coli jm109 strain were used for transformation through heat shock method ( 17 ) . the transformed bacteria were left for 1 hr in lb broth at 37c for recovery , and later 100l of the transformation culture was plated onto an ampicillin ( 100mg / ml ) ( sigma , louis , mo , usa ) , iptg ( sigma ) ( 0.5 mm ) and x - gal ( sigma ) , ( 80g / ml ) containing lb agar plate and was cultured for 16hrs at 37c . the recombinant plasmids were isolated from confirmed colonies by miniperp kit ( qiagen ) and then digested by ecori and noti restriction enzymes . plasmid dna ( 800ng ) was used in 25l of the total volume , including noti ( 15units ) , ( invitrogen , carlsbad , ca , usa ) , ecori ( 15 units ) , ( invitrogen ) , and 2.5l of 10 x reaction buffer 3 ( invitrogen ) and incubated for 1.5 hr at 37c . the mtex101 fragment in pgem - t easy was extracted form agarose gel and then subcloned into a pet-28a ( + ) expression vector ( merck , darmstadt , germany ) and was digested by the same restriction enzymes above . ligation reaction was performed by 125ng of digested pet-28a ( + ) , 0.5pmol of mtex101 purified fragments , 2l of rapid 2x ligation buffer ( pro - mega ) , and 3 units of t4 dna ligase ( promega ) in 10l of the total volume and it was incubated overnight at 4c . ligation products were used for transformation of e. coli jm109 strain by heat shock method ( 17 ) . after a recovery time of 1 hour in lb broth , the transformed bacteria were cultured on lb agar and kanamycin ( 50g / ml ) ( sigma , usa ) containing plates for 16 hrs at 37c . the obtained colonies were screened by colony pcr and one of them was confirmed by dna sequencing . recombinant plasmid was purified and was then used for transformation of bl21 ( de3 ) , which is a protease deficient strain of e. coli . a single transformed colony was inoculated into 50ml of lb broth , including kanamycin ( 50g / ml ) ( sigma , usa ) and untransformed bl21 ( de3 ) bacteria were cultured into 50 ml of lb medium . incubation was performed under shaking at 37c until the optical density ( od ) at 600 nm reached 0.6 . iptg ( sigma ) was added to the remainder with the final concentration of 1 mm and incubation continued for 2 - 3 more hours . cell lysates were prepared by sonication of pellets in pbs buffer including 1% protease inhibitor ( roche ) . protein solutions were obtained from bacteria by boiling them in a sample buffer that contained tris - hcl ( ph=6.8 , 0.5 m ) , sds ( 10% w / v ) , glycerol ( 50% v / v ) , and bromo phenol blue ( 0.5% w / v ) , for 5min . samples ( 50l of each ) were separated by sds - page ( 8% ) ( bio - rad , hercules , ca , usa ) under non - reducing conditions . the protein constituents were then electro - phoretically blotted onto a polyvinylidene difluoride ( pvdf ) membrane ( millipore , billerica , ma , usa ) , as described by towbin et al . 
 ( 13 ) . the membrane was blocked in 5% skim milk / 0.1% tween 20 in pbs ( ph 7.4 ) , and then the reactivity of the transferred protein(s ) with 1g / ml of anti mtex101 pab ( avicenna research institute , iran ) was assessed using sheep anti rabbit ig hrp ( avicenna research institute , iran ) . finally , the membranes were visualized using an ecl system ( ge healthcare , biotech , bucking - hamshire , uk ) . after cloning mtex101 fragments in pgem - t easy vector , several white colonies with probable target fragment inclusion were screened by colony pcr ( figure 2 ) . a right - sized pcr product and a pet-28a(+ ) vector were cut by noti and ecori restriction enzymes to obtain the required fragments for the next step ( figures 3 and 4 ) . coloy pcr on transformed jm109 clones by pgem - t easy vector carrying mtex101 gene . 1 - 5 : 5 selected white colonies , 6 : negative control ( blue colony ) , 7 : positive control ( pcr product on testis cdna ) , 8 : dna ladder viii ( roche ) . double digestion of pgem - t easy vector containing mtex101 fragment with ecori and noti restriction enzymes . 1 : digested vector with mtex101 750bp insert cut out of the vector , 2 : dna ladder viii . double digestion of pet-28a ( + ) expression vector with restriction enzymes . 1 : digested pet-28a ( + ) with ecori and noti , 2 : uncut pet-28a ( + ) , 3 : 1 kb dna ladder . e. coli j m109 strains were transformed using recombinant pet-28a(+ ) containing mtex101 fragment and the undertaken transformation was verified by colony pcr ( figure 5 ) . one of the confirmed colonies ( clone 2 in figure 5 ) was picked for detailed analysis by dna sequencing . alignment of dna sequencing results with mtex101 orf by genbee site ( 14 ) confirmed the cloned sequence . the verified fragment was used for the next round of transformation using e - coli bl21 ( de3 ) strain . colony pcr on transformed jm109 clones by pet-28a ( + ) vector containing mtex101 fragment . 1 and 2 represent two selected colonies , 3 : dna ladder viii , 4 : negative control ( no dna ) , 5 : positive control ( pgem - t easy vector containing mtex101 fragment ) . western blot analysis using anti - mte x 101 peptide polyclonal antibody revealed the correct size of mtex101 recombinant protein production ( 27kda ) in bl-21 ( de3 ) bacteria ( figure 6 ) . western blot analysis of production of the recombinant mtex101 in bl21 ( de3 ) bacteria . 1 : bl21 ( de3 ) containing mtex101 gene after induction , 2 : bl21 ( de3 ) containing mtex101 gene before induction , 3 : untransfected bl21 ( de3 ) , 4 : protein marker ( see blue - invitrogen ) . after cloning mtex101 fragments in pgem - t easy vector , several white colonies with probable target fragment inclusion were screened by colony pcr ( figure 2 ) . a right - sized pcr product and a pet-28a(+ ) vector were cut by noti and ecori restriction enzymes to obtain the required fragments for the next step ( figures 3 and 4 ) . coloy pcr on transformed jm109 clones by pgem - t easy vector carrying mtex101 gene . 1 - 5 : 5 selected white colonies , 6 : negative control ( blue colony ) , 7 : positive control ( pcr product on testis cdna ) , 8 : dna ladder viii ( roche ) . double digestion of pgem - t easy vector containing mtex101 fragment with ecori and noti restriction enzymes . 1 : digested vector with mtex101 750bp insert cut out of the vector , 2 : dna ladder viii . double digestion of pet-28a ( + ) expression vector with restriction enzymes . 1 : digested pet-28a ( + ) with ecori and noti , 2 : uncut pet-28a ( + ) , 3 : 1 kb dna ladder . e. coli j m109 strains were transformed using recombinant pet-28a(+ ) containing mtex101 fragment and the undertaken transformation was verified by colony pcr ( figure 5 ) . one of the confirmed colonies ( clone 2 in figure 5 ) was picked for detailed analysis by dna sequencing . alignment of dna sequencing results with mtex101 orf by genbee site ( 14 ) confirmed the cloned sequence . the verified fragment was used for the next round of transformation using e - coli bl21 ( de3 ) strain . colony pcr on transformed jm109 clones by pet-28a ( + ) vector containing mtex101 fragment . 1 and 2 represent two selected colonies , 3 : dna ladder viii , 4 : negative control ( no dna ) , 5 : positive control ( pgem - t easy vector containing mtex101 fragment ) . western blot analysis using anti - mte x 101 peptide polyclonal antibody revealed the correct size of mtex101 recombinant protein production ( 27kda ) in bl-21 ( de3 ) bacteria ( figure 6 ) . western blot analysis of production of the recombinant mtex101 in bl21 ( de3 ) bacteria . 1 : bl21 ( de3 ) containing mtex101 gene after induction , 2 : bl21 ( de3 ) containing mtex101 gene before induction , 3 : untransfected bl21 ( de3 ) , 4 : protein marker ( see blue - invitrogen ) . the researchers immunized female mice with 8-week old testis lysates and obtained 12 monoclonal antibody producing clones ( tes101 - 112 ) . immunohisto - chemistry analyses revealed that only tes101 was able to recognize a novel testicular protein as determined by searching the expressed sequence tag ( est ) database ( 3 ) . all studies on mtex101 protein are performed solely by tes101 antibody and presently , no commercial antibodies are available to carry out research on this protein . in this study , we produced mtex101 recombinant protein that is useful for antibody development for further investigations . mtex101 mrna contains 750 bases , which encodes 250 amino acids . after the removal of the putative 25-amino acid signal peptide at n - terminus reported a 38kda band by western blot analysis under non - reducing conditions and no 24 kda band was observed ( 3 ) . this large difference in the molecular mass was speculated to be due to glycosylation of the peptide and it was proved to be true by jin et al . glycosylation is known as the prime cause of post - translational modifications ( ptm ) in proteins ( 16 ) . the asn - x - ser / thr is the basic sequence for n - linked glycosylation , however , the secondary structure of the protein can affect the final addition . no consensus sequence has been established for o - linked glycosylation yet ( 17 ) . jin et al , found four putative sites for n - glyco - sylation and several possible sites for o - glyco - sylation in mtex101 amino acid sequence ( 15 ) . in fact , mtex101 is a highly glycosylated protein and it has been clarified that most of the oligosaccharide chains on this peptide are n - linked carbohydrates ( 12 ) . de - n - glycosylation of mtex101 created an 20kda band which is close to or even smaller than estimated molecular weight of mtex101 amino acid backbone ( 15 ) , confirming glycosylation as the cause for the higher mass of native mtex101 . the cellular role of glycoprotein sugar components can be investigated via several methods , like protein production in a host lacking oligo - saccharide addition system , such as prokaryotes ( 18 ) . we succeeded to produce mtex101 recombinant protein in a prokaryotic system , which may be useful in clarifying the role of sugar components in protein structures . however , proteins which are produced by this method may vary in their structures and functions ( 19 ) . western blot analysis showed that this protein had a molecular mass of approximately 27kda under non - reducing conditions that is concordant with the various post - translational modification processes in prokaryotic systems as compared to those in eukaryotes . the size of the recombinant protein ( 27kda ) is in conformity with the backbone protein ( 24kda ) and a 3kda peptide derived from the digested vector by noti and ecori restriction enzymes . in this study , we successfully cloned mtex101 into a his - tagged expression vector ( pet-28a(+ ) ) , followed by an efficient production of the relevant recombinant protein . this protein can be used in antibody production to find out the role of mtex101 in spermatogenesis , egg fertilization and further investigation about the critical role of glycosylation in the function of this protein .