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Molecular Biology Reports

, Volume 46, Issue 2, pp 1593–1601 | Cite as

Construction of a synthetic protein using PCR with a high essential amino acid content for nutritional purposes

  • Ma. I. Sánchez-Crisóstomo
  • M. I. Rojo-López
  • A. Sharma
  • J. C. Cancino-Diaz
  • H. Jaimes-Díaz
  • J. A. Ariza-Ortega
  • E. Madrigal-Santillán
  • G. Betanzos-CabreraEmail author
Original Article
  • 106 Downloads

Abstract

Ovalbumin is considered a protein of high nutritional value because it contains essential amino acids and is highly digestible. Therefore, it has a high biological value. Currently, the high food demand requires worldwide attention because food production is insufficient. Therefore, other alternatives are necessary to satisfy food demands, such as protein engineering. In this work, a protein with a high essential amino acid content similar to ovalbumin was synthesized by protein engineering, expressed, and digested in vitro. The assembly and sequential overlap extension PCR strategy was used to synthesize a 345-bp gene that encodes a high essential amino acid content protein (HEAAP). The 345-bp product was cloned into the vector pBAD TOPO®, and expressed in Escherichia coli BL21. PCR reactions and sequencing demonstrated the presence, orientation, and correct sequence of the insert. HEAAP expression was induced by l-arabinose and then purified using Ni–NTA affinity chromatography. The expression in E. coli was low and barely detected by Western blot assay. The in vitro multienzyme digestibility of HEAAP was around 79%, which suggests that the protein is potentially nutritious. Virtual analysis classifies the protein as unstable and hydrophilic, with a half-life in E. coli of 10 h. The recombinant HEAAP was successfully synthesized, but it is necessary to improve the digestibility and to optimize expression including selecting other expression models.

Keywords

Protein Cloning Expression Digestibility PCR High-value nutritional 

Notes

Acknowledgements

Financial support for this work was provided by CONACYT Problemas Nacionales PDCPN2013-01 and PAI 2015 UAEH.SALUD-2010-01-142035. Juan Carlos Cancino-Díaz is fellow from COFAA-IPN, EDI-IPN and SNI-CONACYT. We also want to thank Fatima A Navarro Gress for its assistance in editing this work.

Conflict of interest

The authors declare they have no conflict of interests regarding the publication of this paper.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Ma. I. Sánchez-Crisóstomo
    • 1
  • M. I. Rojo-López
    • 1
  • A. Sharma
    • 2
  • J. C. Cancino-Diaz
    • 3
  • H. Jaimes-Díaz
    • 4
  • J. A. Ariza-Ortega
    • 1
  • E. Madrigal-Santillán
    • 5
  • G. Betanzos-Cabrera
    • 1
    • 5
    • 6
    Email author
  1. 1.Área Académica de Nutrición, Instituto de Ciencias de la SaludUniversidad Autónoma del Estado de HidalgoSan Agustín TlaxiacaMexico
  2. 2.Tecnologico de Monterrey, School of Engineering and Sciences Campus QueretaroQueretaroMexico
  3. 3.Laboratorio de Inmunomicrobiología. Departamento de MicrobiologíaEscuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional11340 Mexico CityMexico
  4. 4.Laboratory of Biotechnology and Genomic Bioinformatics, Department of Biochemistry, National School of Biological SciencesNational Polytechnic InstituteMexico CityMexico
  5. 5.Área Académica de Medicina, Instituto de Ciencias de la SaludUniversidad Autónoma del Estado de HidalgoSan Agustín TlaxiacaMexico
  6. 6.Universidad Autónoma del Estado de HidalgoPachucaMexico

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