Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1)

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.

Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter

“…2a, lanes 4-6) and was inversely proportional to the inducer concentration. This phenomenon, previously described in the case of other transporters [23], is in agreement with the toxicity of the protein of interest towards bacterial cells. Thus, the lower IPTG concentration resulting in a slower rate of protein production has the effects of decreasing the toxic effect.…”

“…Following IPTG addition, an OD reduction was observed in any condition with the maximum effect at the highest IPTG concentration tested (Fig. 1), highlighting a putative toxic effect of the protein on cell growth, as previously observed for other proteins [23]. Whereas, in the case of pCOLD I, the cells were harvested at a single time since growth was performed at low temperature, as suggested for this speci c plasmid, leading to a very slow rate of growth in all conditions.…”

“…Starting from the insoluble fraction of BL21 cell lysate obtained in optimal conditions, i.e., 8h of induction with 0.05 mM IPTG in presence of 1% glucose at 28°C, the puri cation of hCT2 protein was attempted under denaturing conditions followed by an on-column refolding procedure, as previously described for other SLC transporters [23,24] with substantial modi cations. For refolding, the strong detergent sarkosyl was substituted by octaethylene glycol monododecyl ether (C 12 E 8 ).…”

Strategies for Successful Over-Expression of Human Membrane Transport Systems Using Bacterial Hosts: Future Perspectives

“…The members of the SLC1 family possess eight membrane-spanning domains, are glycosylated, and contain between 524 and 574 amino acids. Even though the 3D-structures of the last two members have recently been solved by Cryo-EM using P. pastoris as the expression host [ 15 , 153 ], the ASCT2 protein was also produced in E. coli [ 53 ]. The expression strategy employed the use of the Rosetta-gami2 strain, which joins the human tRNA supply ( Section 3.1 ) and is involved in disulfide formation ( Section 3 ).…”

“…The serotonin transporter (SLC6A4) was expressed and targeted to E. coli membranes by combining codon optimization and tRNA supply in different strains and media [ 57 ]. Another member of the SLC6 family, the SLC6A19 (also named B0AT1), was expressed in E. coli exploiting human tRNA supplementation (BL21 CodonPlus RIL strain), combining a cold shock strategy (csp promoter, see SLC1A5) with a very low inducer concentration (10 μM) in the presence of 0.5% glucose [ 53 ]. This protein is in complex with ACE2, constituting part of the receptor for the SARS-CoV-2 RBD proteins [ 162 ].…”