Cloning and over Expression Studies of Ovine Somatotropin cDNA of Kajli (sheep breed) in a Prokaryotic System

agri-Abstract: Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-β-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).

contribution of Pakistan to gross domestic product GDP is twenty-one percent with an annual increment of around 2.7 percent 2 . Livestock is an imperative sub-sector of agri-Abstract: Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-β-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).
Key words: cloning, over expression, cDNA, ovine somatotropin, Kajli sheep, prokaryotic system 2 culture in Pakistan making substantial contribution of almost 56 value in addition to the agriculture sector and approximately 11 to GDP 3,4 . Sheep is an important ruminant animal in Pakistan which is typically kept as livestock 5 . It has substantial share in milk 36000 tons , meat 1170000 tons , and Lamb skin 3.08 million production 6 . Due to rapid increase in human population and modernized living style, where people more likely depend upon fast food; meat and milk demands are increasing regularly.
Various conventional methods like cross breeding, appropriate livestock and animal food management strategies have been tried to cope with this increasing demand but still the issue is not solved. Therefore, recombinant DNA technology comes up with a new hope to cover all these concerns. Genetic engineering approaches have provided a great boost in the dairy industry with large scale production of recombinant bovine somatotropin rbST 7 . Production of rbST was one of the earliest agricultural biotechnology-based products to be approved by the U.S. Food and Drug Administration. Somatotropin plays a crucial homeorhetic control in regulating nutrient partitioning. Its introduction to dairy animals increases milk production with improved efficiency of milk synthesizing process by repatriating of glucose to the mammary glands 8 . It works by modifying the gene expression of glucose transporters, particularly in the mammary glands, omental fat and skeletal muscles of respective animals 9 .
In the dairy industry, enhancing productivity is explained as the milk output per feed source input and plays pivotal role in the reduction of natural resource consumption and environmental impact by reducing animal wastage and feed production expenditures. Lower rbST doses during transition and early lactation periods causes improved dry matter intake and body weight provoking healthy alterations in the concentrations of insulin, somatotropin, insulin like growth factor-1, non-esterified fatty acids, calcium and glucose leading to improved milk yield 10 .
Therefore, keeping in view the above background, the current study has been designed to clone somatotropin cDNA gene from a sheep breed, kajli and expressed in bacteria using pLEX expression system. The cloned gene works by altering gene expression of glucose transporters in the animal s mammary gland, skeletal muscle, and omental fat. It facilitates the repartitioning of glucose to the mammary gland, which in turn produces more milk and quality meat production.

Cloning and ligation of oGH cDNA in T/A cloning vector
The study was approved by the ethical committee of GC University Lahore GCUL , Pakistan. Sheep oGH cDNA in T/A vector with signal peptide was cloned and sequenced following the method reported by Khalid and colleagues 11 . PCR purified product was ligated in a T/A cloning vector with manufacturer s protocol InsT/Aclone TM PCR product cloning kit by Fermentas #K1214 . The ligation mixture comprises of 3 µL InsT/A clone TM Vector, 6 µL PCR product, 3 µL ligase buffer 10X , 3 µL PEG 400 solution, 1 µL T4 DNA ligase and deionized water to make total volume up to 30 µL. The ligation mixture was incubated overnight at 22 in the water bath.
Chemically competent cells were i.e. B10 and BL21 DE3 pLysS were prepared 12 . The plates were screened after the incubation for the positive clones. The isolated single colonies were picked and inoculated in 3 mL amp LB broth and incubated at 37 with continuous shaking. The minipreps for plasmid were performed for confirmation.

Restriction analysis and purification of restricted
products After preparation of plasmid from transformed colonies, restriction analysis was carried out to confirm the oGH cDNA presence. The plasmids were restricted with NdeI and HindIII and electrophoresed on 1.2 agarose gel 13 . Restricted DNA fragments representing oGH cDNA in T/A cloning vector were purified from gel with DNA gel extraction kit Novagen, Canada . The purified restricted fragment of oGH cDNA was ligated in the prokaryotic expression vector pET23b Novagen USA , which was also purified after restriction 13,14 . After colonies screening, plasmid was extracted from grown liquid cultures and confirmed using restriction analysis 14 .

Protein expression studies
The clones, confirmed positive by the restriction analysis, were used for protein expression studies. The starter culture of BL21 DE3 pLysS expression host strain having pET23b vector with oGH cDNA, was prepared by inoculating 3 mL of LB broth having ampicillin and chloramphenicol with a single colony. After inoculation the culture was left overnight at 37 with shaking at 200 rpm. From this overnight grown culture 500 µL was added to 50 mL of LB having ampicillin and incubated at 37 with continuous shaking till the OD 600 reached to 0.5. The culture was divided into different tubes and was induced with different isopropyl-L-thio-β D-galactopyronoside IPTG concentrations i.e. 0.1 mM to 0.8 mM; the uninduced cells were used as control. The tubes were left at 37 with continuous shaking at 200 rpm. After optimizing IPTG concentration the experiment was conducted to optimize the induction time. To optimize induction time, the induced culture was taken out at 0, 2, 3, 4 and 5 hrs. The uninduced culture tubes were also taken as control at each induction time. The cells were harvested from the culture by centrifuga-3 tion at 12000 rpm for 20 minutes, supernatant was discarded, and pellet was stored at 80 till further use. The cellular localization of expressed proteins was determined by inducing cells at 24 , 30 and 37 . Proteins were electrophorized by SDS-PAGE with 15 polyacrylamide gel at 250 V for 3 hours. Furthermore, dot blot technique was performed to study expression analysis of desired proteins and optimization of antibodies concentration. During this method, a mixture of proteins is separated based on the basis of molecular weight, and thus various types are separated through gel electrophoresis. The unbound antibody is washed off leaving only the bound antibody to the protein of interest.

Revival of T/A clones and PCR ampli cation
The full length pTZ57R oGH accession no EU074162 clones of oGH cDNA of sheep breed Kajli were revived along with plasmid extraction having signal peptide Fig.  1a . Signal peptide was removed via PCR using specifically designed primers. The forward and reverse primers were designed to remove the signal peptide and amplify gene without stop codon respectively Fig. 1b . Successful elution and purification of PCR product from agarose gel of oGH cDNA is shown in Fig. 1c. 3.2 Cloning of oGH cDNA in T/A cloning vector and restriction analysis After confirmation, amplified and purified PCR product without signal peptide was ligated in a pTZ57R Fermentas ® Lithuania UAB cloning vector afterwards its transfor- mation in chemically competent E. coli B10 strain. After transformation, these cells were plated on LB agar plates having antibiotic ampicillin and left in incubated overnight at 37 13 . Next day isolated colonies were cultured in 2-5 mL liquid LB medium containing 50 µg/mL ampiciline and left in shaking incubator overnight at 37 . Plasmid extraction from overnight grown culture and gel electrophoresis confirmed successful cloning by showing plasmid having insert of 3459 bp Fig. 2a . After harvesting the plasmids from potential colonies, restriction enzymes NdeI and HindIII were used for analysis. Interestingly, these enzyme sites were present in primers used for amplification through PCR. The restricted plasmids were electrophoresed on agarose gel and showed a fragment insert of 600 bps confirming the clone as positive having oGH cDNA Fig. 2b . Subsequently, presence of restricted insert was purified and confirmed by gel electrophoresis 1.2 agarose gel as shown in Fig. 2c.

Revival of pET23b prokaryotic expression vector;
puri cation, ligation and transformation Ovine GH was sub-cloned into pET23b expression vector with a strong T7 promoter which was then transformed into E. coli strain BL21 DE3 . Glycerol stocks of bacterial plasmid expression vectors pET23b containing cloned cGH gene was used to get pET23b vector minus cGH for cloning of oGH cDNA. Therefore, said clone was cultured in 3 mL of LB containing appropriate antibiotic. Overnight grown liquid cultures of cells were used to isolate recombinant plasmid and run on 1 agarose gel to make sure the presence of recombinant plasmid Fig. 2d . Afterwards, extracted pET plasmid having cloned cGH was restricted with NdeI and HindIII Fig. 2e .
To clone oGH cDNA in pET23b , restricted product of plasmid was eluted and confirmed by gel electrophoresis Fig. 2f . The purified oGH cDNA was used for ligation with restricted and purified pET23b vector. The expression strains derived from BL21 DE3 pLysS were transformed with recombinant plasmid vectors. After overnight incubation of transformed bacterial cells, single colonies from each plate were picked and inoculated in appropriate antibiotic supplemented LB broth. Recombinant plasmid having oGH pEToGH were isolated from transformed cells of BL21 DE3 pLysS and electrophoresed on 1 agarose gel Fig. 2g . The results confirmed the transformation as size of pET23b increased with insert. Presence of insert was further confirmed by restriction analysis of isolated recombinant plasmids by NdeI and HindIII enzyme Fig. 2h 13 .

Protein expression analysis
The restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors have an IPTG inducible strong T7 promoter and E. coli expression strain of BL21 DE3 pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various concentrations of IPTG for the optimization of expression levels. Expression strain BL21 DE pLysS harboring pET oGH were grown in Amp and Cam supplemented LB broth until OD 600 reached 0.5, where maximum cells were in logarithmic phase of growth. Cells were induced with different IPTG concentrations 0.1 to 0.8 mM followed by SDS-PAGE. Results indicated maximum expression level of oGH at concentration of 0.3 mM. Further gel result indicated molecular weight of oGH to be 22 kDa Fig. 3a . Once the finest IPTG concentration was determined, the expression level was standardized. Figure  3b illustrates the SDS-PAGE expression results of oGH with time gradient. Maximum expression was observed at 5 hrs after induction of cells with 0.3 mM IPTG concentration. To check the cellular localization of protein, cell pellet was sonicated discontinuously and resulting proteins were divided in to 5 groups i.e. supernatant, total cytoplasmic proteins TCP or dissolved proteins and pellet, inclusion bodies IB or aggregating proteins. Accumulation of expressed oGH in IBs was shown in Fig. 3c. Moreover, dot blot analysis was performed for confirmation of expressed proteins with IPTG concentration and time gradient optimized samples Fig. 3d .

Discussion
The present study showed the cloning and expression of ovine growth hormone from a local sheep breed, Kajli in a prokaryotic system for the first time in Pakistan. Livestock is an important sector of agriculture in Pakistan. Among several other animals, sheep is one of the vital domesticated animals in Pakistan. It forms an important and fundamental part of small owner agriculture. Pakistan has great variety of indigenous sheep breeds which are divided into fat tail and thin tail breeds 15 . The domestic ovine species are vital source of meat mutton and milk predominantly in the rural areas of the country 16 . Sheep milk is an important unprocessed material for the milk industry principally in cheese manufacturing 17 . All sheep breeds in Pakistan yield coarse type wool and are largely raised in assorted flocks together with goats.
Moreover, sheep is a central animal to be sacrificed and considered as an important animal to many societies as it fits well to diverse socio-economic and environmental settings 18 . With an expanding human population and increasing standards of living, rapidly increased demand for milk and meat was reported. Since past, man is trying to cope with this increasing demand by several conventional methods like proper livestock management, cross breeding and animal feed management. Biotechnology has been heralded as a science that has an innovatory impact on agriculture. Growth hormone GH which can now be produced by biotechnology, have massive impact on dairy industry 19,20 .
Therefore, due to the importance of GH in meat and milk production, its cDNA has been cloned from a local sheep breed, Kajli and expressed in bacteria using pET expression system. Various other studies have used the bacterial system for growth hormone expression 21,22 . The administration of exogenous GH, a non-glycosylated polypeptide hormone, has been exposed to increase lactation and performance in mammals not only in laboratory animals but in humans too 21 . Farmers are using GH for substantial increase in milk and have shown enormous applications in goats, pigs, cows and sheep 24 .

Conclusion
In conclusion, the results of the current study showed that cloning and over expression of somatotropin cDNA play very immense role in the growth of sheep. It stimulates the growth of bone and cartilage. GH boosts protein production, promotes the utilization of fat, interferes with the action of insulin, and help in milk and wool production within a shorter period of time. Cloning and expression of GH cDNA in bacteria is expected to make a revolutionary change in the history of biotechnology to boost up the economy of any country through dairy industry. It also opens the way to find out the alternative techniques to enhance the production of growth hormone.

Conflict of Interests
On behalf of all authors, the corresponding author states that there is no conflict of interest.

Author's contribution
IL Corresponding author designed and supervised study. MST performed experiment and wrote the manuscript. SZ, NF, NM, IL facilitated to perform different techniques during research experiment. GS, FR and AB helped in graphical aspects and data analysis. IL revised and ap-