Expression of the Biologically Active Insulin Analog SCI-57 in Nicotiana Benthamiana

Adriana Muñoz-Talavera; Miguel Ángel Gómez-Lim; Luis A Salazar-Olivo; Jörg Reinders; Katharina Lim; Abraham Escobedo-Moratilla; Alberto Cristian López-Calleja; María Cristina Islas-Carbajal; Ana Rosa Rincón-Sánchez

doi:10.3389/fphar.2019.01335

Expression of the Biologically Active Insulin Analog SCI-57 in Nicotiana Benthamiana

Front Pharmacol. 2019 Nov 14:10:1335. doi: 10.3389/fphar.2019.01335. eCollection 2019.

Authors

Adriana Muñoz-Talavera¹, Miguel Ángel Gómez-Lim², Luis A Salazar-Olivo³, Jörg Reinders⁴, Katharina Lim⁵, Abraham Escobedo-Moratilla⁶, Alberto Cristian López-Calleja², María Cristina Islas-Carbajal¹, Ana Rosa Rincón-Sánchez⁷

Affiliations

¹ Department of Physiology, Institute of Experimental and Clinical Therapeutics, University Center for Health Sciences, University of Guadalajara, Guadalajara, Mexico.
² Department of Genetic Engineering, Center for Research and Advanced Studies of the National Polytechnic Institute, Irapuato, Mexico.
³ Division of Molecular Biology, Institute for Scientific and Technological Research of San Luis Potosí, San Luis Potosí, Mexico.
⁴ Scientific Support Unit Analytical Chemistry, Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany.
⁵ Institute of Functional Genomics, University of Regensburg, Regensburg, Germany.
⁶ CONACYT-Consortium for Research, Innovation, and Development of the Drylands (CIIDZA), IPICYT, San Luis Potosí, Mexico.
⁷ Institute of Molecular Biology and Gene Therapy, Department of Molecular Biology and Genomic, University Center for Health Sciences, University of Guadalajara, Guadalajara, Mexico.

Abstract

Diabetes mellitus is a growing problem worldwide; however, only 23% of low-income countries have access to insulin, and ironically it costs higher in such countries than high-income ones. Therefore, new strategies for insulin and insulin analogs production are urgently required to improve low-cost access to therapeutic products, so as to contain the diabetes epidemic. SCI-57 is an insulin analog with a greater affinity for the insulin receptor and lower thermal degradation than native insulin. It also shows native mitogenicity and insulin-like biological activity. In this work, SCI-57 was transiently expressed in the Nicotiana benthamiana (Nb) plant, and we also evaluated some of its relevant biological effects. An expression plasmid was engineered to translate an N-terminal ubiquitin and C-terminal endoplasmic reticulum-targeting signal KDEL, in order to increase protein expression and stability. Likewise, the effect of co-expression of influenza M2 ion channel (M2) on the expression of insulin analog SCI-57 (SCI-57/M2) was evaluated. Although using M2 increases yield, it tends to alter the SCI-57 amino acid sequence, possibly promoting the formation of oligomers. Purification of SCI-57 was achieved by FPLC cation exchange and ultrafiltration of N. benthamiana leaf extract (NLE). SCI-57 exerts its anti-diabetic properties by stimulating glucose uptake in adipocytes, without affecting the lipid accumulation process. Expression of the insulin analog in agroinfiltrated plants was confirmed by SDS-PAGE, RP-HPLC, and MS. Proteome changes related to the expression of heterologous proteins on N. benthamiana were not observed; up-regulated proteins were related to the agroinfiltration process. Our results demonstrate the potential for producing a biologically active insulin analog, SCI-57, by transient expression in Nb.

Keywords: 3T3-L1 adipocytes; Nicotiana benthamiana; diabetes; insulin analog SCI-57; proteomic profile; transient expression.