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Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1).
Galluccio, Michele; Pantanella, Marta; Giudice, Deborah; Brescia, Stefania; Indiveri, Cesare.
  • Galluccio M; 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.
  • Pantanella M; 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.
  • Giudice D; 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.
  • Brescia S; 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.
  • Indiveri C; 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. cesare.indiveri@unical.it.
Mol Biol Rep ; 47(9): 7283-7289, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-705288
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Pneumonia, Viral / Minor Histocompatibility Antigens / Coronavirus Infections / Amino Acid Transport Systems, Neutral / Amino Acid Transport System ASC / Escherichia coli / Betacoronavirus Limits: Humans Language: English Journal: Mol Biol Rep Year: 2020 Document Type: Article Affiliation country: S11033-020-05717-8

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Pneumonia, Viral / Minor Histocompatibility Antigens / Coronavirus Infections / Amino Acid Transport Systems, Neutral / Amino Acid Transport System ASC / Escherichia coli / Betacoronavirus Limits: Humans Language: English Journal: Mol Biol Rep Year: 2020 Document Type: Article Affiliation country: S11033-020-05717-8