Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization Beatriz Nogueira Messias Miranda a,b , Wesley Luzetti Fotoran c , Fernanda Canduri a , Dulce Helena Ferreira Souza d , Gerhard Wunderlich c , Emanuel Carrilho a,b,* a Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, 13566-590 São Carlos, SP, Brazil b Instituto Nacional de Ciência e Tecnologia de Bioanalítica - INCTBio, 13083-970, Campinas, SP, Brazil c Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374 - Cidade Universitária, São Paulo, SP, Brazil d Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, s/n, São Carlos, SP, Brazil ARTICLE INFO Keywords: Alpha folate receptor Trigger factor Expression E. coli Exosomes ABSTRACT The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time- consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylpho- sphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straight- forward and versatile approach for the production of active human FRα by heterologous expression; this ap- proach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors. 1. Introduction Folate receptors (FRs) are glycosyl-phosphatidylinositol (GPI)-an- chored proteins that bind folic acid (folate) and its reduced derivate with high affinity [1]. Its role in folate metabolism and cancer devel- opment has been extensively studied, particularly for the α isoform. The expression of folate receptor 1 (FOLR1 or FRα) is restricted to the apical surface of polarized epithelial cells of specific tissues [2–4], playing an important role in the normal development of neurons. FRα is crucial during early fetal development [5], with special implications for neu- rological disorders and physiopathology of tumor development. Over- expression of FRα was confirmed in lung [6–8], kidney [3], and gy- necological adenocarcinomas [1,9–13] demonstrating not only its direct relation to disease development but also its potential use as a highly sensitive and specific biomarker for cancer therapies [1,4,8]. Recently, an additional role for FRα was reported. FRα appears to activate oncogenic transcription factors [5,14] related to malformations of the embryo and neurological syndromes [5], suggesting the existence of other unknown functions. To explore the structural information of folate receptors, Wibowo et al. (2013) and Chen et al. (2013) de- termined the crystal structure of human FRα in complex with different ligands [15,16] using mammalian expression systems. Unfortunately, this fabrication process required a multi-step, complex process that showed two substantial disadvantages: high cost and time consuming [17]. Alternatively, the cheaper heterologous expression of human FRα in E. coli system would promote relative simplicity, and shorter culti- vation time, sparking significant interest for the antifolate testing de- velopment. Until now, no report has focused specifically on FRα het- erologous expression due to the incorrect folding, thus inactive structure, of this protein, when produced in E. coli. [18]. Here we present an efficient system for the expression and pur- ification of an FRα in E. coli, as shown in Fig. 1. Unlike a conventional expression method, we use a protein fusion to express the target protein together with a trigger factor (TF)—a highly soluble ribosome-asso- ciated chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains—enhancing its solubility [18–22]. We http://dx.doi.org/10.1016/j.pep.2017.10.006 Received 17 August 2017; Received in revised form 3 October 2017; Accepted 3 October 2017 * Corresponding author. Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, 13566-590 São Carlos, SP, Brazil. E-mail address: emanuel@iqsc.usp.br (E. Carrilho). Protein Expression and Purification 142 (2018) 75–80 Available online 05 October 2017 1046-5928/ © 2017 Elsevier Inc. All rights reserved. MARK