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BMC Proceedings

Open Access

The influence of Escherichia coli cultivation temperature on interferon alpha 2a expression (IFN-α2a)

  • Fernanda dos Santos Arthuso1,
  • Miriam Fussae Suzuki1,
  • Nélio Alessandro de Jesus Oliveira2,
  • João Ezequiel de Oliveira1,
  • Paolo Bartolini1 and
  • Carlos Roberto Jorge Soares1
BMC Proceedings20148(Suppl 4):P2

https://doi.org/10.1186/1753-6561-8-S4-P2

Published: 1 October 2014

Background

The Hormone Group of IPEN develops researches on recombinant pituitary hormones produced in Escherichia coli at laboratory scale, including human growth hormone (hGH) and prolactin (hPRL). The best results were obtained using the λPL promoter and the signal peptide DsbA, and so the protein of interest is secreted into the bacterial periplasmic space, in its authentic form without an initial methionine [1, 2]. The expression, under control of the λPL promoter, regulated by the thermosensitive repressor, is used for large scale production in E. coli. Thermal induction presents a great advantage over chemical inducers like IPTG or nalidixic acid, that are expensive and dangerous for manipulators and environment, but it presents the disadvantage that proteins particularly thermolabile like hPRL could suffer proteolysis and aggregation, influencing negatively the production. Considering the limitations on the use of IPTG and the periplasmic secretion, successfully obtained with our hGH expression vector using λPL promoter and W3110 strain, we decided to use this system for IFN-α2a production. The goal of this work was the construction of the IFN-α2a vector, obtaining an the IFN-α2a periplasmic expression.

Methods

The DNA sequences corresponding to the NdeI restriction site, start codon, signal peptide DsbA, cDNA of the IFN-α2a, stop codon, and the restriction site BamHI were inserted into the plasmid, λPL-DsbA-mPRL [3]. This plasmid contains the λPL promoter and the gene for ampicillin resistance [1]. The new vector was called λPL-DsbA-IFNα2a. The IFN-α2a sequence was synthesized by the GenScript Corporation (Piscataway - NJ - EUA) in the pUC57 plasmid. The vector obtained was amplified in E. coli DH5α strain and then introduced into the expression W3110 cells. After selection, the best clone was used to test different cultivation temperatures (32°C, 35°C, 37°C, and 42°C). Analyses of the periplasmic fluid obtained by osmotic shock based on Western Blotting and SDS-PAGE were carried out.

Results and conclusions

Construction of λPL-DsbA-IFNα2a plasmid was confirmed by restriction analysis. IFN-α2a secreted into the periplasmic space of E. coli was obtained, as shown in Western Blotting and SDS-PAGE. We can observe that the best growth temperature for the IFN-α2a was 37°C probably because at this temperature the bacteria presented a better growth and the production of aggregates was lower. The production yield was of ~ 0,1 µg/ml/A600.

Declarations

Acknowledgements

This work was supported by FAPESP, São Paulo (Project n. 2007/59540-3) and by CNPq, Brasília (Projects GD 141221/2011-9, PQ 303839/2008-2, PQ 3000473/2009-5 and DT 310512/2010/7).

Authors’ Affiliations

(1)
Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP
(2)
Lillehei Heart Institute, University of Minnesota

References

  1. Soares CR, Gomide FI, Ueda EK, Bartolini P: Periplasmic expression of human growth hormone via plasmid vectors containing the lambdaPL promoter: use of HPLC for product quantification. Protein Engineering. 2003, 16 (12): 1131-8.View ArticlePubMedGoogle Scholar
  2. Soares CR, Ueda EK, Oliveira TL, Gomide FI, Heller SR, Bartolini P: Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields. Journal of Biotechnology. 2008, 133 (1): 27-35.View ArticlePubMedGoogle Scholar
  3. Suzuki MF, Arthuso FS, Oliveira JE, Oliveira NA, Goulart HR, Capone MV, Ribela MT, Bartolini P, Soares CR: Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space. Biotechnology and Applied Biochemistry. 2012, 59 (3): 178-85.View ArticlePubMedGoogle Scholar

Copyright

© Santos Arthuso et al.; licensee BioMed Central Ltd. 2014

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.

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