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Production and purification of recombinant glargine insulin from Escherichia coli BL-21 strain

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Abstract

Diabetes is a chronic metabolic disorder that has affected approximately 463 million populations till date and this number is expected to exponentially rise in the coming decade. Majority of diabetic patients require multiple insulin injections regularly, at the risk of hypoglycemia. As an alternative to conventional insulin, glargine insulin, a long-acting insulin analog that helps in maintaining blood glucose levels, is being preferred. Hence, the present work focuses on glargine insulin production using recombinant E. coli BL-21(DE3) cells through fed-batch fermentation. After fermentation, the cells were lysed through high pressure homogenization to release inclusion bodies containing insulin glargine precursor and the major impurities like Arg (B31)-insulin were reduced by incorporating citraconylation in appropriate concentration. These steps are novel which makes the present study distinct from the other. Furthermore, refolding and enzymatic digestion of insulin glargine precursor was carried out to obtain glargine insulin. Consequently, purification and polishing of glargine insulin were performed by loading onto cation exchange chromatography and reverse-phase high performance liquid chromatography which fetched 98.6% pure product. Through the aforementioned process, from 20 L of culture broth, nearly 0.3 g/L of recombinant glargine insulin with high purity was obtained in two purification steps. Hence, the present study devises an efficient and economical process for large-scale production of glargine insulin.

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Data availability

The data that support the findings of this study are recorded in the manuscript and any further information will be made available on request from the first author.

The authors express their consent for communicating this manuscript to your journal and moreover confirm that this article has not been communicated to any other journal for publication.

Abbreviations

Gly:

Glycine

Ile:

Isoleucine

Val:

Valine

Glu:

Glutamic acid

Gln:

Glutamine

Cys:

Cysteine

Thr:

Threonine

Ser:

Serine

Leu:

Leucine

Tyr:

Tyrosine

Asn:

Asparagine

Phe:

Phenylalanine

His:

Histidine

Ala:

Alanine

Arg:

Arginine

Pro:

Proline

Lys:

Lysine

Da:

Dalton

References

  1. E. Haythorne, M. Rohm, M. van de Bunt, M.F. Brereton, A.I. Tarasov, T.S. Blacker, F.M. Ashcroft, Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells. Nat Commun 10(1), 1–17 (2019)

    Article  Google Scholar 

  2. C.F. Deacon, Dipeptidyl peptidase 4 inhibitors in the treatment of type 2 diabetes mellitus. Nat Rev Endocrinol 16(11), 642–653 (2020)

    Article  CAS  Google Scholar 

  3. M.J. Kim, S.H. Min, S.Y. Shin, M.N. Kim, H. Lee, J.Y. Jang, H.S. Jung, Attenuation of PERK enhances glucose-stimulated insulin secretion in islets. J Endocrinol 236, 125–136 (2018)

    Article  CAS  Google Scholar 

  4. T.N. Vinther, M. Norrman, U. Ribel, K. Huss, M. Schlein, D.B. Steensgaard, F. Hubalek, Insulin analog with additional disulfide bond has increased stability and preserved activity. Protein Sci 22, 296–305 (2013)

    Article  CAS  Google Scholar 

  5. Y. Kollati, R.R. Ambati, P.N. Reddy, N.S.S. Kumar, R.K. Patel, V.R. Dirisala, Congenital hypothyroidism: facts, facets & therapy. Curr Pharm Des 23(16), 2308–2313 (2017)

    Article  CAS  Google Scholar 

  6. S. Khan, M.W. Ullah, R. Siddique, G. Nabi, S. Manan, M. Yousaf, H. Hou, Role of recombinant DNA technology to improve life. International Journal of Genomics, 2016(2405954), 14 (2016)

    Google Scholar 

  7. R. Patra, Y. Kollati, N.S. Sampath Kumar, V.R. Dirisala, Valsartan in combination with metformin and gliclazide in diabetic rat model using developed RP-HPLC method. Future J Pharma Sci 7(1), 1–10 (2021)

    Article  Google Scholar 

  8. P. Hazra, S. Sreenivas, K. Venkatesan, M.B. Patale, A. Chatterjee, N. Ramprabu, M. Kusumanchi, A novel peptide design aids in the expression and its simplified process of manufacturing of Insulin Glargine in Pichia pastoris. Appl Microbiol Biotechnol. 105, 3061–3074 (2021)

    Article  CAS  Google Scholar 

  9. A. Lapolla, M.G. Dalfra, Hundred Years of Insulin Therapy: Purified Early Insulins. Am J Ther 27, e24–e29 (2020)

    Article  Google Scholar 

  10. X. Zhao, H. Zong, A. Abdulla, E.S. Yang, Q. Wang, J.Y. Ji, F. Yang, Inhibition of SREBP transcriptional activity by a boron-containing compound improves lipid homeostasis in diet-induced obesity. Diabetes 63, 2464–2473 (2014)

    Article  Google Scholar 

  11. G.E. Umpierrez, D.C. Klonoff, Diabetes technology update: use of insulin pumps and continuous glucose monitoring in the hospital. Diabetes Care 41, 1579–1589 (2018)

    Article  CAS  Google Scholar 

  12. P. Sonksen, J. Sonksen, Insulin: understanding its action in health and disease. Br J Anaesth 85(1), 69–79 (2000)

    Article  CAS  Google Scholar 

  13. H.G. Hwang, K.J. Kim, S.H. Lee, C.K. Kim, C.K. Min, J.M. Yun, S.U. Lee, Y.J. Son, Recombinant glargine insulin production process using Escherichia coli. J Microbiol Biotechnol 26, 1781–1789 (2016)

    Article  CAS  Google Scholar 

  14. A. Bera, S. Herbert, A. Jakob, W. Vollmer, F. Götz, Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol Microbiol 55, 778–787 (2005)

    Article  CAS  Google Scholar 

  15. R. Patra, P.N. Reddy, N.S. Kumar, V.R. Dirisala, Novel Validated RP-HPLC Method for Simultaneous Estimation of Valsartan & Gliclazide in Bulk and Dosage Forms. Curr Pharm Anal 14(4), 412–418 (2018)

    Article  CAS  Google Scholar 

  16. R.A. Nazeer, R. Kavitha, R.J. Ganesh, S.Y. Naqash, N.S. Kumar, R. Ranjith, Detection of collagen through FTIR and HPLC from the body and foot of Donax cuneatus Linnaeus, 1758. J Food Sci Technol 51(4), 750–755 (2014)

    Article  CAS  Google Scholar 

  17. E.J. Bennett, N.F. Bence, R. Jayakumar, R.R. Kopito, Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell 17, 351–365 (2005)

    Article  CAS  Google Scholar 

  18. G.L. Rosano, E.A. Ceccarelli, Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5, 172 (2014)

    Google Scholar 

  19. M.M. Carrio, A. Villaverde, Role of molecular chaperones in inclusion body formation. FEBS Lett 537, 215–221 (2003)

    Article  CAS  Google Scholar 

  20. A. Singh, V. Upadhyay, A.K. Upadhyay, S.M. Singh, A.K. Panda, Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Factories 14, 1–10 (2015)

    Article  CAS  Google Scholar 

  21. A. Sarker, A.S. Rathore, R.D. Gupta, Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process. Microb Cell Factories 18, 5 (2019)

    Article  Google Scholar 

  22. K. Ashutosh, K.A. Dinesh, K. Sunil, Y. Mohan Reddy, A.D. Chintagunta, K.V. Saritha, P. Govind, S.P. Jeevan Kumar, Nutraceuticals derived from seed storage proteins: Implications for health wellness. Biocatal Agric Biotechnol 17, 710–719 (2019)

    Article  Google Scholar 

  23. P. Singhvi, A. Saneja, S. Srichandan, A.K. Panda, Bacterial inclusion bodies: a treasure trove of bioactive proteins. Trends Biotechnol 38, 474–486 (2020)

    Article  CAS  Google Scholar 

  24. N. Venkata Raju, K. Sukumar, G.R. Babul, P.K. Pankaj, G. Muralitharan, A. Shanti, D. Teja Sai, A.D. Chintagunta, In-Vitro Studies on Antitumour and Antimicrobial Activities of Methanolic Kernel Extract of MangiferaIndica L. Cultivar Banganapalli. Biomed Pharmacol J 12(1), 357–362 (2019)

    Article  Google Scholar 

  25. C.K. Kim, S.B. Lee, Y.J. Son, Large-scale refolding and enzyme reaction of human preproinsulin for production of human insulin. J Microbiol Biotechnol 25(10), 1742–1750 (2015)

    Article  CAS  Google Scholar 

  26. H.K.S. Leiros, B.O. Brandsdal, O.A. Andersen, V. Os, I. Leiros, R. Helland, A.O. Smalas, Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements. Protein Sci 13, 1056–1070 (2004)

    Article  CAS  Google Scholar 

  27. Y. Xie, S. Min, N.P. Harte, H. Kirk, J.E. Obrien, H.P. Voorheis, K. Hun Mok, Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex A study using charge‐specific chemical modifications. Proteins: Struct Funct Bioinf 81, 1–17 (2013)

    Article  CAS  Google Scholar 

  28. M. Thevis, A. Thomas, W. Schanzer, Mass spectrometric determination of insulins and their degradation products in sports drug testing. Mass Spectrom Rev 27, 35–50 (2008)

    Article  CAS  Google Scholar 

  29. T. Arakawa, K. Tsumoto, K. Nagase, D. Ejima, The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. Protein Expr Purif 54, 110–116 (2007)

    Article  CAS  Google Scholar 

  30. M.Weiss, D.F. Steiner, L.H. Philipson, Insulin biosynthesis, secretion, structure, and structure-activity relationships (2015)

  31. D. LeRoith, H. Werner, D. Beitner-Johnson, C.T. Roberts, Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16, 143–163 (1995)

    Article  CAS  Google Scholar 

  32. N.S. Sampath Kumar, S. NorizahMhd, S.R. Sandeep, A.D. Chintagunta, S. Prathibha, I. Satheesh Kumar, S.P. Jeevan Kumar, B. Sai Anvesh, D. VijayaRamu, Extraction of bioactive compounds from Psidium guajava leaves and its utilization in preparation of jellies. AMB Express 11, 36 (2021)

    Article  CAS  Google Scholar 

  33. M. Zielinski, A. Romanik-Chruscielewska, D. Mikiewicz, N. Lukasiewicz, I. Sokolowska, J. Antosik, A. Plucienniczak, Expression and purification of recombinant human insulin from E. coli 20 strain. Protein Expr Purif 157, 63–69 (2019)

    Article  CAS  Google Scholar 

  34. K. Govender, T. Naicker, J. Lin, S. Baijnath, A.A. Chuturgoon, N.S. Abdul, T. Docrat, H.G. Kruger, T. Govender, A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli). AMB Express 10, 1–9 (2020)

  35. S. Polez, D. Origi, S. Zahariev, C. Guarnaccia, S.G. Tisminetzky, N. Skoko, M. Baralle, A simplified and efficient process for insulin production in Pichia pastoris 11, e0167207 (2016)

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Acknowledgements

This is a part of the PhD work carried by the first author SBK. The first author gratefully acknowledges the support extended by Nanogen Biopharmaceutical, Vietnam, for providing no objection certificate to execute the PhD work along with publishing articles. In addition to it, help extended by Vcare Biolabs Pvt. Ltd., Hyderabad, for slight improvement of the work is also acknowledged.

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Authors and Affiliations

  1. Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research, Vadlamudi, Andhra Pradesh, 522213, India

    Satish Babu Kaki, Anjani Devi Chintagunta, N. S. Sampath Kumar, Vijaya R. Dirisala & M. Sai Krishna

  2. Nanogen Biopharmaceutical, Ho Chi Minh, Vietnam

    Satish Babu Kaki

  3. Wockhardt Research Centre, Aurangabad, 431210, Maharashtra, India

    A. Naga Prasad

  4. Vcare Biolabs Pvt. Ltd., Telangana, 500090, Hyderabad, India

    S. J. K. Naidu & B. Ramesh

Authors
  1. Satish Babu Kaki
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  2. Anjani Devi Chintagunta
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  3. A. Naga Prasad
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  4. N. S. Sampath Kumar
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  5. Vijaya R. Dirisala
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  6. M. Sai Krishna
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  7. S. J. K. Naidu
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  8. B. Ramesh
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Contributions

Satish Babu Kaki: designed and executed the work and drafted the manuscript; Anjani Devi Chintagunta: reviewing the manuscript; A. Naga Prasad: supported in executing the work; N.S. Sampath Kumar: valuable insights during periodic reviews of the work and helped in drafting the manuscript; Vijaya R. Dirisala: reviewing the manuscript, M. Sai Krishna: provided inputs in drafting the manuscript; S.J.K. Naidu and B. Ramesh: supported with resources.

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Correspondence to N. S. Sampath Kumar.

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Kaki, S.B., Chintagunta, A.D., Prasad, A.N. et al. Production and purification of recombinant glargine insulin from Escherichia coli BL-21 strain. emergent mater. 5, 335–346 (2022). https://doi.org/10.1007/s42247-021-00313-3

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  • DOI: https://doi.org/10.1007/s42247-021-00313-3

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