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-Cabrera

Research output: Contribution to journalArticle

Abstract

© 2019, Springer Nature B.V. 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.
Original languageAmerican English
JournalMolecular Biology Reports
DOIs
StatePublished - 1 Jan 2019

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