Secretory expression of human insulin precursor in Pichia pastoris using truncated α-factor leader sequence and a short C-peptide

https://doi.org/10.22146/ijbiotech.38958

Dini Nurdiani(1*), Hariyatun Hariyatun(2), Wien Kusharyoto(3)

(1) Research Center for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Bogor Km. 46, Cibinong, 16911, Indonesia
(2) Research Center for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Bogor Km. 46, Cibinong, 16911, Indonesia
(3) Research Center for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Bogor Km. 46, Cibinong, 16911, Indonesia
(*) Corresponding Author

Abstract


In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. Increasing number of diabetic patients led to the escalation of recombinant insulin demand which was creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It had excellent qualifications as an expression host in large-scale production of recombinant proteins for therapeutic use. We attempted in this study to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in a frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones have been successfully obtained and verified using PCR with AOX1 specific primers for the integration of the expression cassette into P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by P. pastoris clone was confirmed by SDS-PAGE of the culture supernatant. It showed a single band of the secreted IP with a molecular mass above 6.5 kDa.


Keywords


diabetes, recombinant human insulin precursor, P. pastoris, α-factor and C-peptide.



References

Ahmad M, Hirz M, Pichler H, Schwab H. 2014. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 98 (12):5301-5317. doi:10.1007/s00253-014-5732-5.

Baeshen MN, Bouback TAF, Alzubaidi MA, Bora RS, Alotaibi MAT, Alabbas OTO, Alshahrani SM, Aljohani AAM, Munshi RAA, Al-Hejin A, et al. 2016. Expression and purification of C-Peptide containing insulin using Pichia pastoris expression system. BioMed Res Intl. 2016. doi:10.1155/2016/3423685.

Baeshen NA, Baeshen MN, Sheikh A, Bora RS, Ahmed MMM, Ramadan HAI, Saini KS, Redwan EM. 2014. Cell factories for insulin production. Microb Cell Fact. 13(141). doi:10.1186/s12934-014-0141-0.

Cregg JM, Vedvick TS, Raschke WC. 1993. Recent advances in the expression of foreign genes in Pichia pastoris. BioTechnology. 11(8):905-910. doi:10.1038/nbt0893-905.

Fu Z, Gilbert ER, Liu D. 2013. Regulation of insulin synthesis and secretion and pancreatic beta-cell dysfunction in diabetes. Curr Diabetes Rev. 9(1):25-53. doi:10.2174/157339913804143225.

Gurramkonda C, Polez S, Skoko N, Adnan A, Gäbel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U. 2010. Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Fact. 9(31). doi:10.1186/1475-2859-9-31.

Haider SR, Reid HJ, Sharp BL. 2012. Tricine SDS-PAGE. Methods Mol Biol. 869:81-91. doi:10.1007/978-1-61779-821-4_8.

Summer H, Grämer R, Dröge P. 2009. Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE). J Vis Exp. (32): 1485. doi: 10.3791/1485.

Invitrogen. 2010. EasySelect™ Pichia Expression Kit for expression of recombinant proteins using pPICZ and pPICZα in Pichia pastoris.

Juturu V, Wu JC. 2018. Heterologous protein expression in Pichia pastoris: latest research progress and applications. Chem Bio Chem. 19(1):7-21. doi:10.1002/cbic.201700460.

Kjeldsen T, Ludvigsen S, Diers I, Balschmidt P, Sørensen AR, Kaarsholm NC. 2002. Engineering-enhanced protein secretory expression in yeast with application to insulin. J Biol Chem. 277(21):18245-18248. doi:10.1074/jbc.C200137200.

Kjeldsen T, Pettersson AF, Hach M. 1999. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem. 29(1):79-86. doi:10.1111/j.1470-8744.1999.tb01151.x.

Kjeldsen T. 2000. Yeast secretory expression of insulin precursors. Appl Microbiol Biotechnol. 54(3):277-286. doi:10.1007/s002530000402.

Kjeldsen TB, Ludvigsen S. 2012. Method for making insulin precursors and insulin precursors analogues having improved fermentation yield in yeast. U.S. Patent US8129146B2.

Kurjan J, Herskowitz I. 1982. Structure of a yeast pheromone gene (IMFα): a putative α-factor precursor contains four tandem copies of mature α-factor. Cell. 30(3):933-943. doi:10.1016/0092-8674(82)90298-7.

Lin-Cereghino GP, Stark CM, Kim D, Chang J, Shaheen N, Poerwanto H, Agari K, Moua P, Low LK, Tran N, et al. 2013. The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene. 519(2):311-317. doi:10.1016/j.gene.2013.01.062.

Meehl MA, Stadheim TA. 2014. Biopharmaceutical discovery and production in yeast. Curr Opin Biotechnol. 30:120-127. doi:10.1016/j.copbio.2014.06.007.

Nilsson J, Jonasson P, Samuelsson E, Stahl S, Uhlén M. 1996. Integrated production of human insulin and its C-peptide. J Biotechnol. 48(3):241-250. doi:10.1016/0168-1656(96)01514-3.

Ogurtsova K, da Rocha Fernandes JD, Huang Y, Linnenkamp U, Guariguata L, Cho NH, Cavan D, Shaw JE, Makaroff  LE. 2017. IDF diabetes atlas: global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract. 128:40-50. doi:10.1016/j.diabres.2017.03.024.

Polez S, Origi D, Zahariev S, Guarnaccia C, Tisminetzky SG, Skoko N, Baralle M. 2016. A simplified and efficient process for insulin production in Pichia pastoris. PloS One. 11(12):e0167207. doi:10.1371/journal.pone.0167207.

Porro D, Sauer M, Branduardi P, Mattanovich D. 2005. Recombinant protein production in yeasts. Mol Biotechnol. 31(3):245-259. doi:10.1385/MB:31:3:245.

Sambrook J, Russel D. 2001. Molecular cloning: a laboratory manual. 3rd ed. New York (NY): Cold Spring Harbor Laboratory Press.

Stern B, Olsen L, Trosse C, Ravneberg H, Pryme I. 2007. Improving mammalian cell factories: the selection of signal peptide has major impact on recombinant protein synthesis and secretion in mammalian cells. Trends Cell Mol Biol. 2:1-17.

Thim L, Hansen MT, Norris K, Hoegh I, Boel E, Forstromt J, Ammerertt G, Fiil NP. 1986. Secretion and processing of insulin precursors in yeast. Proc Natl Acad Sci USA. 83(18):6766-6770. doi:10.1073/pnas.83.18.6766.

Vassileva A, Chugh DA, Swaminathan S, Khanna N. 2001. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter. J Biotechnol. 88(1):21-35. doi:10.1016/S0168-1656(01)00254-1.

Wang Y, Liang ZH, Zhang YS, Yao SY, Xu YG., Tang YH, Zhu SQ, Cui DF, Feng YM. 2001. Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product. Biotechnol Bioeng. 73(1):74-79. doi:10.1002/1097-0290(20010405)73:1<74::AID-BIT1038>3.0.CO;2-V.

[WHO] World Health Organization. 2016. Global Report on Diabetes. [cited 2018 Aug 10]. Available from: http://www.who.int/diabetes/global-report/en/

Xie T, Liu Q, Xie F, Liu H, Zhang Y. 2008. Secretory expression of insulin precursor in Pichia pastoris and simple procedure for producing recombinant human insulin. Prep Biochem Biotechnol. 38(3):308-317. doi:10.1080/10826060802165147.



DOI: https://doi.org/10.22146/ijbiotech.38958

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