Expression,purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli |
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| Institution: | 1. Instituto Politécnico Nacional-CIBA, Ex-Hacienda San Juan Molino Carretera estatal Tecuexcomac-Tepetitla Km 1.5, 90700 Tepetitla, Tlaxcala, Mexico;2. Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados de IPN, Libramiento Norte Carretera Irapuato León Kilómetro 9.6, Carretera Irapuato León, 36821 Irapuato, Guanajuato, Mexico;3. Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F., Mexico |
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| Abstract: | BackgroundThe acidic subunit of amarantin (AAC)—the predominant amaranth seed storage protein—has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4).ResultsE. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves.ConclusionsModifications to the third VR affect the thermal stability of amarantin acidic subunit. |
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