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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 53 issue 5 (may 2019) : 587-593,   Doi: 10.18805/ijar.B-3571

Expression and characterization of recombinant single chain beta-alpha equine chorionic gonadotropin in prokaryotic host 

A
Anuradha Bhardwaj
S
Sanjay Kumar
V
Varij Nayan
P
Parvati Sharma
Y
Yash Pal
S
S.C. Yadav
1ICAR-National Research Centre on Equines, Sirsa Road, Hisar-125 001, Haryana, India.
Cite article:- Bhardwaj Anuradha, Kumar Sanjay, Nayan Varij, Sharma Parvati, Pal Yash, Yadav S.C. (2018). Expression and characterization of recombinant single chain beta-alpha equine chorionic gonadotropin in prokaryotic host. Indian Journal of Animal Research. 53(5): 587-593. doi: 10.18805/ijar.B-3571.

ABSTRACT

The aim of present study was to produce recombinant eCG/pituitary glycoprotein in the Escherichia coli (E. coli) BL21C host cells and to test its diagnostic efficacy. This aim was achieved by optimizing its expression, purification as well as its characterization through the immunoassays and bioassays. A bacterial protein expression vector system based on the phage T7 promoter and histidine tag was used for the expression and purification. The recombinant single chain beta-alpha equine chorionic gonadotropin (rbaeCG) encoding gene was constructed with beta and alpha sequences according to its biologically active counterpart. It was successfully cloned and when expressed in E.coli BL21C host, the purified recombinant protein was found to be active as revealed by enzyme linked immunosorbent assay (ELISA) and Western blotting. However, it was not found to exhibit any significant activity in vivo when tested in the mice

REFERENCES

  1. Allen WR, Moor RM (1972). The origin of the equine endometrial cups. I. Production of PMSG by fetal trophoblast cells. J Reprod Fertil. 29:313-316.
  2. Bhardwaj A, Nayan V, Yadav P, De S, Datta TK, Goswami SL (2012). Heterologous expression and characterization of Indian Sahiwal Cattle (Bosindicus) alpha inhibin. Anim Biotechnol 23:71-88
  3. Bhardwaj, A., Nayan, V., De, S., & Goswami, S. L. (2013). Differential expression profiling of recombinant bovine Inhibin-alpha at reduced temperature. Ind J of Anim Res, 47(1), 61–65.
  4. Bhardwaj, A, Nayan, V., Sharma, P. Sanjay Kumar, Yash Pal, Jitender Singh. (2017). Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix. In Silico Pharmacol. 5:5. https://doi.org/10.1007/s40203-017-0025-1. 
  5. Bousfield GR, Butnev VY (2001). Identification of twelve O-glycosylation sites in equine chorionic gonadotropin beta and equine luteinizing hormone ss by solid-phase Edman degradation. Biol Reprod 64:136-147.
  6. Cole HH, Erway J (1941). 48-Hour assay test for equine gonadotropin with results expressed in international units. Endocrinology 29:514-519. 
  7. Combarnous Y (1992). Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocr Rev 13:670-691.
  8. Coulibaly S, Besenfelder U, Miller I, Zinovieva N, Lassnig C, Kotler T, Jameson JL, Gemeiner M, Muller M, Brem G (2002). Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSH alpha/beta) produced in the milk of transgenic rabbits. Mol Reprod Dev 63:300-308.
  9. Fidler AE, Lun S, Young W, McNatty KP (1998). Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris. J Mol Endocrinol 21:327-336.
  10. Galet C, Chopineau M, Martinat N, Combarnous Y, Guillou F (2000). Expression of an in vitro biologically active equine LH/CG without C-terminal pepetide and/or B-26-110 disulphide bridge. J Endocrinol 167:117-124.
  11. Galet C, Le Bourhis CM, Chopineau M, Le Griec G, Perrin A, Magallon T, Attal J, Viglietta C, Houdebine LM, Guillou F (2001). Expression of a single beta alpha chain protein of equine LH/ CG in milk of transgenic rabbits and its biological activity. Mol Cell Endocrinol 174:31-40.
  12. Koles K, van Berkel PHC, Mannesse MLM, Zoetemelk R, Vliegenthart JFG, Kamerling JP (2004). Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits. Glycobiology 14:979-986.
  13. LeãoRde B and Esteves SC (2014). Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech. Clinics (Sao Paulo) 69:279-93.
  14. Legardinier S, Duonor-Cerutti M, Devauchelle G, Combarnous Y, Cahoreau C (2005). Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines. J Mol Endocrinol 34: 47-60.
  15. Legardinier S, Poirier JC, Klett D, Combarnous Y, Cahoreau C (2008). Stability and biological activities of heterodimeric and single-    chain equine LH/chorionic gonadotropin variants. J Mol Endocrinol 40:185-98.
  16. Min KS, Shinozaki M, Miyazawa K, Nishimura R, Sasaki N, Shiota K and Ogawa (1994). Nucleotide sequence of eCG a subunit cDNA and its expression in the equine placenta. J Reprod Dev 40:301-305.
  17. Müller C, Wagner K, Frankenfeld K, Franzreb M (2011). Simplified purification of equine chorionic gonadotropin (eCG)-an example of the use of magnetic microsorbents for the isolation of glycoproteins from serum. Biotechnol Lett 33:929-36. 
  18. Rosano GL, Ceccarelli EA (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol 5:172.
  19. Sambrook J et al. (2000). Molecular Cloning: A Laboratory Manual (Third Edition)
  20. Seo EB, Kang MH, Min KS (2015). Biological activities of tethered recombinant equine chorionic gonadotropin mutants (bGln94Alaa, bIle95Alaa and bLys96Alaa). Academia Journal of Biotechnology 3(6):122-131.
  21. Sherman GB, Wolfe MW, Farmerie TA, Clay CM, Threadgill DS, Sharp DC, Nilson JH (1992). A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin. Mol Endocrinol 6:951-959.
  22. Towbin H, Staehelin T, Gordon J (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 76(9):4350-4.
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Indian Journal of Animal Research
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    Research Article

    volume 53 issue 5 (may 2019) : 587-593,   Doi: 10.18805/ijar.B-3571

    Expression and characterization of recombinant single chain beta-alpha equine chorionic gonadotropin in prokaryotic host 

    A
    Anuradha Bhardwaj
    S
    Sanjay Kumar
    V
    Varij Nayan
    P
    Parvati Sharma
    Y
    Yash Pal
    S
    S.C. Yadav
    1ICAR-National Research Centre on Equines, Sirsa Road, Hisar-125 001, Haryana, India.
    Cite article:- Bhardwaj Anuradha, Kumar Sanjay, Nayan Varij, Sharma Parvati, Pal Yash, Yadav S.C. (2018). Expression and characterization of recombinant single chain beta-alpha equine chorionic gonadotropin in prokaryotic host. Indian Journal of Animal Research. 53(5): 587-593. doi: 10.18805/ijar.B-3571.

    ABSTRACT

    The aim of present study was to produce recombinant eCG/pituitary glycoprotein in the Escherichia coli (E. coli) BL21C host cells and to test its diagnostic efficacy. This aim was achieved by optimizing its expression, purification as well as its characterization through the immunoassays and bioassays. A bacterial protein expression vector system based on the phage T7 promoter and histidine tag was used for the expression and purification. The recombinant single chain beta-alpha equine chorionic gonadotropin (rbaeCG) encoding gene was constructed with beta and alpha sequences according to its biologically active counterpart. It was successfully cloned and when expressed in E.coli BL21C host, the purified recombinant protein was found to be active as revealed by enzyme linked immunosorbent assay (ELISA) and Western blotting. However, it was not found to exhibit any significant activity in vivo when tested in the mice

    REFERENCES

    1. Allen WR, Moor RM (1972). The origin of the equine endometrial cups. I. Production of PMSG by fetal trophoblast cells. J Reprod Fertil. 29:313-316.
    2. Bhardwaj A, Nayan V, Yadav P, De S, Datta TK, Goswami SL (2012). Heterologous expression and characterization of Indian Sahiwal Cattle (Bosindicus) alpha inhibin. Anim Biotechnol 23:71-88
    3. Bhardwaj, A., Nayan, V., De, S., & Goswami, S. L. (2013). Differential expression profiling of recombinant bovine Inhibin-alpha at reduced temperature. Ind J of Anim Res, 47(1), 61–65.
    4. Bhardwaj, A, Nayan, V., Sharma, P. Sanjay Kumar, Yash Pal, Jitender Singh. (2017). Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix. In Silico Pharmacol. 5:5. https://doi.org/10.1007/s40203-017-0025-1. 
    5. Bousfield GR, Butnev VY (2001). Identification of twelve O-glycosylation sites in equine chorionic gonadotropin beta and equine luteinizing hormone ss by solid-phase Edman degradation. Biol Reprod 64:136-147.
    6. Cole HH, Erway J (1941). 48-Hour assay test for equine gonadotropin with results expressed in international units. Endocrinology 29:514-519. 
    7. Combarnous Y (1992). Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocr Rev 13:670-691.
    8. Coulibaly S, Besenfelder U, Miller I, Zinovieva N, Lassnig C, Kotler T, Jameson JL, Gemeiner M, Muller M, Brem G (2002). Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSH alpha/beta) produced in the milk of transgenic rabbits. Mol Reprod Dev 63:300-308.
    9. Fidler AE, Lun S, Young W, McNatty KP (1998). Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris. J Mol Endocrinol 21:327-336.
    10. Galet C, Chopineau M, Martinat N, Combarnous Y, Guillou F (2000). Expression of an in vitro biologically active equine LH/CG without C-terminal pepetide and/or B-26-110 disulphide bridge. J Endocrinol 167:117-124.
    11. Galet C, Le Bourhis CM, Chopineau M, Le Griec G, Perrin A, Magallon T, Attal J, Viglietta C, Houdebine LM, Guillou F (2001). Expression of a single beta alpha chain protein of equine LH/ CG in milk of transgenic rabbits and its biological activity. Mol Cell Endocrinol 174:31-40.
    12. Koles K, van Berkel PHC, Mannesse MLM, Zoetemelk R, Vliegenthart JFG, Kamerling JP (2004). Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits. Glycobiology 14:979-986.
    13. LeãoRde B and Esteves SC (2014). Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech. Clinics (Sao Paulo) 69:279-93.
    14. Legardinier S, Duonor-Cerutti M, Devauchelle G, Combarnous Y, Cahoreau C (2005). Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines. J Mol Endocrinol 34: 47-60.
    15. Legardinier S, Poirier JC, Klett D, Combarnous Y, Cahoreau C (2008). Stability and biological activities of heterodimeric and single-    chain equine LH/chorionic gonadotropin variants. J Mol Endocrinol 40:185-98.
    16. Min KS, Shinozaki M, Miyazawa K, Nishimura R, Sasaki N, Shiota K and Ogawa (1994). Nucleotide sequence of eCG a subunit cDNA and its expression in the equine placenta. J Reprod Dev 40:301-305.
    17. Müller C, Wagner K, Frankenfeld K, Franzreb M (2011). Simplified purification of equine chorionic gonadotropin (eCG)-an example of the use of magnetic microsorbents for the isolation of glycoproteins from serum. Biotechnol Lett 33:929-36. 
    18. Rosano GL, Ceccarelli EA (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol 5:172.
    19. Sambrook J et al. (2000). Molecular Cloning: A Laboratory Manual (Third Edition)
    20. Seo EB, Kang MH, Min KS (2015). Biological activities of tethered recombinant equine chorionic gonadotropin mutants (bGln94Alaa, bIle95Alaa and bLys96Alaa). Academia Journal of Biotechnology 3(6):122-131.
    21. Sherman GB, Wolfe MW, Farmerie TA, Clay CM, Threadgill DS, Sharp DC, Nilson JH (1992). A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin. Mol Endocrinol 6:951-959.
    22. Towbin H, Staehelin T, Gordon J (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 76(9):4350-4.
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