Production of recombinant human xCT (SLC7A11) and reconstitution in proteoliposomes for functional studies

Abstract

The plasma membrane transporter xCT belongs to the SLC7 family and has the physiological role of mediating the exchange of glutamate and cystine across the cell plasma membrane, being crucial for redox control. The xCT protein forms a heterodimer with the ancillary protein CD98. Over the years, xCT became a hot pharmacological target due to the documented over-expression in virtually all human cancers, which rely on cystine availability for their progression. Notwithstanding, several unknown aspects of xCT biology still exist that require a suitable single protein experimental model, to be addressed. To this aim, the recombinant host Escherichia coli has been exploited to over-express the human isoform of xCT. In this widely used and low-cost system, the optimization for growth and protein production has been achieved by acting on the metabolic needs of the bacterial strains. Then, the His-tagged protein has been purified by Ni2+-chelating chromatography and reconstituted in proteoliposomes for transport activity assays. The expressed protein was in a folded/active state allowing functional and kinetic characterization. Interestingly, the features of the recombinant protein meet those of the native one extracted from intact cells, further confirming the suitability of E. coli as a host for the expression of human proteins. This study opens perspectives for elucidating other molecular aspects of xCT, as well as for studying the interaction with endogenous and exogenous compounds, relevant to human health.

Over-Production of the Human SLC7A10 in E. coli and Functional Assay in Proteoliposomes

“…This, together with glucose addition, exerted a positive effect on protein production, probably due to catabolite repression, which prevented the production of the protein in the absence of an inducer, avoiding toxicity problems [ 15 ]. Indeed, a putative toxic effect of the recombinant protein was confirmed by the inverse relationship between the inducer concentration and protein synthesis, as already observed for other amino acid transporters, probably due to Sec-translocon saturation [ 16 , 19 ]. A fundamental role in hASC-1 production was exerted by oxygen availability, which was increased by decreasing the broth volume in each flask.…”

“…To improve the production of the protein of interest, the same E. coli strains were cultured either in the absence or in the presence of 0.5% glucose ( Figure 2 ), since it is known that glucose addition may positively affect the production of human plasma membrane proteins [ 15 , 16 ].…”

“…Furthermore, it cannot be excluded that the presence of oxygen allowed E. coli to produce the human transporter in a condition from which the refolding was easier to perform. Indeed, this was different from other human SLC transporters produced in E. coli , which were refolded on-column, substituting the ionic detergent sarkosyl with a non-ionic detergent [ 16 , 21 ]. hASC-1 could be solubilized directly with the non-ionic C 12 E 8 .…”

“…A fundamental role in hASC-1 production was exerted by oxygen availability, which was increased by decreasing the broth volume in each flask. In this respect, hASC-1 behaved, in E. coli , similarly to another member of the SLC7 family, the SLC7A11 (xCT) transporter [ 16 ]. The dependence on oxygen may be linked to a metabolic load affecting E. coli cells, which positively respond to the increased oxygen availability for overcoming the stress due to the over-expression of a potentially toxic transporter.…”

“…A more suitable procedure for studying the transport in a single protein experimental model is the heterologous production of the human transporter and its reconstitution in proteoliposomes. This approach allows for abolishing interferences by other transport proteins and has been extensively adopted for many SLC transporters [ 15 , 16 , 17 ]. It has been particularly useful for unveiling unknown aspects of the transport mechanism and regulation of SLC25A20 (CAC), SLC1A5 (ASCT2), SLC7A5 (LAT1), SLC22A4 (OCTN1), SLC38A9, SLC7A11 (xCT) [ 15 , 18 ], and many others.…”