Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1)
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Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1) Michele Galluccio1 · Marta Pantanella1 · Deborah Giudice1 · Stefania Brescia1 · Cesare Indiveri1,2 Received: 4 May 2020 / Accepted: 2 August 2020 © Springer Nature B.V. 2020
Abstract It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/ function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5–1%), at low temperature (15 °C), and low inducer concentrations (10– 100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins. Keywords Amino acid · SLC · Protein expression · B0AT1 · ASCT2 · COVID-19
Introduction Escherichia coli represents the most used expression bacterial host, due to its easy handling, low costs, fast growth, and high expression yield. However, in many cases bacteria refuse to express human proteins for several reasons [1]. In these cases, the most frequently adopted strategy is that of switching to yeast or to mammalian cell lines. However, the yield in purified protein obtained with these approaches is often very poor and the cost, especially with mammalian cells, is much higher than the E. coli system [2]. Major * Cesare Indiveri [email protected] 1
Department of DiBEST (Biologia, Ecologia, Scienze Della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata Di Rende, CS, Italy
CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Via Amendola 122/O, 70126 Bari, Italy
2
difficulties in over-expressing human proteins have been encountered in the case of integral membrane proteins, such as transporters, which contain extensive hydrophobic regions and lead to bacterial cell toxicity [3]. This m
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