- Poster presentation
- Open access
- Published:
Recombinant human proinsulin expression in Pichiapastoris using PGK promoter
BMC Proceedings volume 8, Article number: P88 (2014)
Background
There are indications, according to WHO, that diabetes will reach 333 million people in 2025, and already is among one of the main death causes in the world. In 2030 in Latin America, diabetes mellitus will be the second cause of deaths [1]. The actions to increase the production of insulin are strategic to respond the high demands for what we have to face in the near future [2]. P. pastoris methylotrophic yeast is being very used in the last decades to express heterologous genes to biotech uses [3]. The main expression vectors to Pichia pastoris are based in the strong promoter from AOX1 gene which codifies alcohol oxidize enzyme. However there is a search for new promoters than AOX1 due to certain disadvantages as the use of methanol as inducer that is toxic and originates undesirable subproducts during its metabolism [4]. This work has the main objective in to express in high levels the human proinsulin in Pichia pastoris using PGK promoter.
Methods
The human proinsulin codifing sequence was chemically synthesized (Genone inc.) with P. pastoris optimized codons. The synthesized sequence was subcloned under PGK promoter control in expression vector together with the codifying region from Saccaromyces cerevisiae factor-α signal peptide in order to have proinsulin expression/secretion. After linearization the vector containing the expression cassete was introduced in P. pastoris GS115 by electroporation. To select clones, with multicopies of expression cassette, was realized a zeocin resistance test in plate [5]. Resistant clones to 100 μg/mL of zeocin were selected and submitted to crescent concentrations of the antibiotic: 500, 1000 and 2000 μg/mL. The recombinant clones that produced/secreted more proinsulin was detected by imunodot procedure using antibody anti-insulin/proinsulin (Pierce-D3E7/5B6/6). Selected recombinant clones were cultivated in YPD medium for 24 hours in 30°C at 200rpm and the supernatants were clarified by centrifugation and analyzed by ELISA and Western-blotting.
Results and conclusion
From 200 zeocin (100 µg/mL) resistant P. pastoris clones 25 were able to grow in 2000 µg/mL which indicates that they have integrated several expression/ secretion cassetes in their genomes. From those one was notable for its higher human proinsulin expression/secretion in both experiments (imunodot and ELISA). The proinsulin overexpression of this selected clone was confirmed by PAGE and Western-blotting with anti-insulin/anti-proinsulin antibody. Additional experiments are in process in order to standardize the fermentative process for expression and secretion of human proinsulin as well as confirm recombinant protein amino acid sequence.
References
WHO/NCD/NCS/99.2: Definition, diagnosis and classification of diabetes mellitus and its complications. World Health Organization. 1999, Accessed on 02.09.2012, [http://whqlibdoc.who.int/hq/1999/WHO_NCD_NCS_99.2.pdf]
Brand-Miller Jennie: A Nova Revolução da Glicose. Rio de Janeiro: Campus/Elsevier. 2003
Fazenda S, Mcneil ML, Harvey B, L M: Heterologous protein production using the Pichia pastoris expression system. Yeast. 2005, 22: 249-270.
Almeida JRM, Moraes LMP, Torres FAG: Molecular characterization of the phosphoglycerate kinase (PGK1) from the methylotrophic yeast Pichia pastoris. Yeast. 2005, 22: 725-737.
Vassileva A, Chugh DA, Swaminathan S, Khanna N: Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter. J Biotechnol. 2001, 88: 21-35. doi: 10.1016/S0168-1656(01)00254
Acknowledgements
This work was supported by CNPq, CAPES, FAPEAM and FAPDF.
Author information
Authors and Affiliations
Rights and permissions
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
de Assunçao, E.N., Cristina dos, S.M., Araripe, T.F. et al. Recombinant human proinsulin expression in Pichiapastoris using PGK promoter. BMC Proc 8 (Suppl 4), P88 (2014). https://doi.org/10.1186/1753-6561-8-S4-P88
Published:
DOI: https://doi.org/10.1186/1753-6561-8-S4-P88