Expression Analysis of Recombinant Equine Chorionic Gonadotropin in Three Host Systems: E. coli BL21C, Sf insect cell lysate and COS-1 mammalian cells

DOI: 10.18805/ijar.B-3917    | Article Id: B-3917 | Page : 40-45
Citation :- Expression Analysis of Recombinant Equine Chorionic Gonadotropin in Three Host Systems: E. coli BL21C, Sf insect cell lysate and COS-1 mammalian cells.Indian Journal Of Animal Research.2021.(55):40-45
Anuradha Bhardwaj, Varij Nayan, Sanjay Kumar, Parvati Sharma, Sanjeev Kumar, Neha Chakarvarty, Sudarshan Kumar, Yash Pal, S.C. Yadav, A.K. Mohanty, B.N. Tripathi dranu.biotech@gmail.com
Address : ICAR-National Research Centre on Equines, Hisar-125 001, Haryana, India.  
Submitted Date : 4-09-2019
Accepted Date : 25-11-2019

Abstract

Mammalian cells are the recommended host for recombinant eukaryotic protein production aimed at incorporation of post-translational modifications for downstream applications. The bacterial system and insect cells are widely used because of ease of technical methodology, economics of production, purification and yield of final protein. The present research objective was expression of recombinant reproductive hormones of animal origin and study of their immunogenic potential for reproductive applications. The equine Chorionic Gonadotropin (eCG) is one of the most heavily glycosylated protein amongst all glycoprotein hormone family. Hence, experiments were carried out to observe its expression in the three most popular host systems and it led to comparative studies for their post-translational modifications. The Pregnant Mare Serum Gonadotropin (PMSG, also called as eCG) gene was cloned in TOPO-TA vector, pIX 4.0 and pTARGET vectors accordingly and expression analysis in E. coli BL21C, Sf insect cell lysate and COS-1 cells was carried out. We observed diverse sizes of recombinant proteins in SDS-PAGE analysis which indicated post-translational modification in mammalian expression system towards the linking of tags as well as side chains in respective host cells. Basic diagnostic immunogenicity tests showed encouraging results, however, no significant in vivo and in vitro activity was observed for the expressed reCG in all the employed host systems.

Keywords

Equine Gonadotropin Hormone Host Protein Recombinant

References

  1. Allen W.R. and Moor R.M. (1972). The origin of the equine endo-    -metrial cups. I. Production of PMSG by fetal trophoblast cells. Journal of Reproduction and Fertility. 29(2): 313-316.
  2. Bhardwaj A., Kumar S., Nayan V., Sharma P., Pal Y. and Yadav S.C. (2019). Expression and characterization of recombinant single chain beta-alpha equine chorionic gonadotropin in prokaryotic host. Indian Journal of Animal Research. 53(5): 587–593.
  3. Bhardwaj A., Nayan V., De S. and Goswami S.L. (2013). Differential Expression Profiling of Recombinant Bovine Inhibin-Alpha at Reduced Temperature. Indian Journal of Animal Research. 47(1): 61–65.
  4. Bhardwaj A., Nayan V., Parvati, Mamta and Gupta A.K. (2012). Inhibin: A Role for Fecundity Augmentation in Farm Animals. Asian Journal of Animal and Veterinary Advances. 7(9): 771-789.
  5. Bhardwaj A., Nayan V., Sharma P., Kumar S., Pal Y. and J. Singh. (2017). Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix. In Silico Pharmacology. 5(1): 5.
  6. Bhardwaj, A. Nayan V., Yadav P., De S., Datta T.K. and Goswami S.L. (2012). Heterologous Expression and Characteri- -zation of Indian Sahiwal Cattle (Bos indicus) Alpha Inhibin. Animal Biotechnology. 23(2): 71–88.
  7. Combarnous Y. (1992). Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocrine Reviews. 13(4): 670–691.
  8. Faraj N., Alhalabi M. andAl- Quobaili F. (2017). Predictive value of follicular fluid insulin like growth factor-1 in IVF outcome of normo-ovulatory women. Middle East Fertility Society Journal. 22(2): 101–104.
  9. Jeoung Y.H., Yoon J.T. and Min K.S. (2010). Biological Functions of the COOH-Terminal Amino Acids of the á-Subunit of Tethered Equine Chorionic Gonadotropin. Reproductive and Developmental Biology. 34(1): 47–53.
  10. Leão R. de B.F. and Esteves S.C. (2014). Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech. Clinics (Sao Paulo). 69(4): 279–293.
  11. 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. Journal of Molecular Endocrinology. 34(1): 47–60.
  12. Nayan V., Bhardwaj A. and Singh D. (2013). PAPP-A in Indian Water Buffalo (Bubalus bubalis) Ovary: Phylogeny, Expression, Hormonal Regulation and Sequence Characterization. Agricultural Research 2(2): 153–165.
  13. Park J.J., JarGal N., Yoon J.T. and. Min K.S. (2010). â-Subunit 94~96 Residues of Tethered Recombinant Equine Chorionic Gonadotropin are Important Sites for Luteinizing Hormone and Follicle Stimulating Hormone like Activities. Reproductive and Developmental Biology. 34(1): 33–40.
  14. Park J.J., Seong H.K., J.S. Kim, Munkhzaya B., Kang M.H and. Min K.S (2017). Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells. Development and Reproduction. 21(2): 111–120. 

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