Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein Obtained from Rhodococcus erythropolis
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The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with the addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18, immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3). Then, the amplified expression of the DYC18_RS18060-His6 protein was achieved with detection through SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728). It was also predicted to form twelve transmembrane spanning α-helices, with both N- and C-terminal ends at the cytoplasmic side of the membrane. Database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine; however, this was uncertain based on the comparison of residues involved in substrate binding in experimentally characterised NCS-1 proteins. The current study lays the foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins.
Keywords: bioinformatics analysis, gene cloning, membrane topology, NCS-1 family, protein expression, transport protein
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