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Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP

  • Bryan D. Santos
  • Jose Ruben Morones-Ramirez
  • Isaias Balderas-Renteria
  • Nestor G. Casillas-Vega
  • David W. Galbraith
  • Xristo ZarateEmail author
Original paper
  • 11 Downloads

Abstract

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

Keywords

SmbP IMAC Protein expression and purification Periplasm Signal sequence Sec pathway PelB RFP CusF Tat pathway GFP TorA 

Notes

Acknowledgements

We thank Mexico’s Consejo Nacional de Ciencia y Tecnologia (CONACYT) for the financial support provided to BDS during his graduate studies. This work was funded by Grants UANL-PAICYT-2015 CN567-15 and CONACYT Grant CB 2012-179,774-B awarded to XZ.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Universidad Autonoma de Nuevo Leon, Facultad de Ciencias QuimicasSan Nicolas de los GarzaMexico
  2. 2.Universidad Autonoma de Nuevo Leon, Centro de Investigacion en Biotecnologia y Nanotecnologia, Facultad de Ciencias Quimicas, Parque de Investigacion e Innovacion TecnologicaApodacaMexico
  3. 3.Departamento de Patologia ClinicaUniversidad Autonoma de Nuevo Leon, Hospital Universitario Dr. Jose Eleuterio GonzalezMonterreyMexico
  4. 4.School of Plant Sciences and BIO5 Institute, University of ArizonaTucsonUSA

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