Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein from Rhodococcus Erythropolis

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 addition of Eco RI and Pst I restriction sites at the 5′ and 3′ ends, Copyright The Authors. University respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His 6 -tag. The construct was transformed into Escherichia coli BL21 (DE3), then amplified expression of the DYC18_RS18060-His 6 protein was achieved with detection by 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) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. of DYC18_RS18060. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins.

A crucial step in the pipeline for structural and functional characterisation of a membrane protein is overcoming the challenge of achieving amplified expression [29] so that sufficient quantities of protein can be available for crystallisation trials and for applying various chemical, biochemical and biophysical techniques. In the present work we have cloned the Rhodococcus erythropolis gene DYC18_RS18060, which putatively codes for an NCS-1 transporter, and achieved amplified recombinant protein expression in E. coli. We have also performed a bioinformatics analysis of the chemical and physical properties, predicted structure and function characteristics, and evolutionary relationships of the DYC18_RS18060 protein. Whilst DYC18_RS18060 is not itself a drug target, bacterial NCS-1 proteins are close homologues of human LeuT-fold solute carrier transporters [30][31][32][33], which are drug targets for the treatment of disease [34][35][36].

Recombinant Protein Expression
A clone of E. coli BL21(DE3) cells transformed with pTTQ18/DYC18_RS18060-His6 was streaked on to an LB-agar plate (1.5%) containing carbenicillin (Melford Laboratories, UK) (100 µg/mL) and incubated at 37°C overnight. Expression of DYC18_RS18060-His6 was tested from small-scale cultures that were grown in LB medium (50 mL) supplemented with carbenicillin (100 µg/mL). A single colony was used to inoculate the LB medium and the culture was incubated (37°C, 220 rpm) up to an A600 of 0.6. Induction was initiated by adding isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Melford Laboratories, UK) (0.5 mM), then growth was continued for 2 hours before harvesting the cells by centrifugation (12000 x g, 4 °C, 10 minutes). Mixed (inner plus outer) membranes were isolated from the cells using a water lysis procedure. Successful amplified expression of the DYC18_RS18060-His6 protein was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.

Large-Scale Cultures and Membrane Preparation
For large-scale membrane preparation, a total of 10 litres of cells in 2-litre flasks were grown to an A600 of 0.6, then induced with IPTG (0.5 mM) and grown for a further 3 hours before harvesting by centrifugation (6000 x g, 15 min, 4°C) and storage at -80°C. At a later time the cells were thawed, suspended in Tris-EDTA buffer (20 mM Tris, pH 7.5 with 0.5 mM EDTA) and disrupted by passing twice through a cell disrupter (Constant Systems) at 30 kpsi.
Undisrupted cells and cell debris were removed by centrifugation at 12000 x g for 45 minutes at 4°C. The supernatant containing total (inner plus outer) membranes was collected.
Inner/outer membranes were separated by sucrose gradient ultracentrifugation and prepared as described in Ward et al. [38], followed by washing and resuspension in Tris buffer (20 mM, pH 7.5), dispensing into aliquots, freezing in liquid nitrogen and storage at -80°C.

SDS-PAGE and Western Blotting
SDS-PAGE used 4% stacking gels and 15% resolving gels made from acrylamide (40%) and bisacrylamide (2%) solutions (BioRad Laboratories). Samples contained 10 g protein and gels were stained with Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific). For western blotting, samples containing 5 g protein were first separated by SDS-PAGE and then transferred from the gel to a Fluorotrans TM membrane (Pall BioSupport, UK) using a Trans-Blot semi-dry transfer cell (BioRad) operating at 18 volts for 35 minutes. This involved pre-soaking four pieces of filter paper in 0.5x SDS-PAGE running buffer, then two pieces of filter paper followed by the membrane, the polyacrylamide gel and two further pieces of filter paper were layered onto one another. Following transfer, the membrane was incubated with bovine serum albumin (3%) in TBST (20 mM Tris-HCl pH 7.6, 0.05% v/v Tween-20, 0.5M NaCl) for 3 hours at 4°C to block non-specific binding sites. The membrane was washed twice with TBST (20 mL) at room temperature for 10 minutes. The membrane was then incubated for 1 hour with HisProbe-HRP antibody (QIAGEN Ltd) (10 mL) diluted to 1:5000 with TBST followed by three washes with TBST (20 mL) for 10 minutes each. A 6-mL SuperSignal West Pico chemiluminescent solution was prepared by mixing 3 mL West Pico luminol/enhancer solution (Perbio Science, UK) (3 mL) and West Pico stable peroxide solution (Perbio Science, UK) (3 mL) and the membrane was incubated with this for 3 minutes before wrapping in acetate for exposure (Syngene G:Box).

Computational Methods
Gene and protein sequence information was obtained from the National Center for

Cloning and Amplification of the DYC18_RS18060 Gene
The PCR primers designed for cloning and amplifying the DYC18_RS18060 gene from R.
erythropolis with a His6-tag were predicted to be free of dimers or other secondary structures.
They were also predicted to have other ideal properties, including a melting temperature of ≥65 °C, a GC content of less than 40% and termination with a G or C base. Analysis of the PCR product by agarose gel electrophoresis showed that the DYC18_RS18060 gene was successfully cloned and amplified. According to restriction digestion analysis of the plasmid construct, the DYC18_RS18060 gene had been successfully ligated into pTTQ18 at the EcoRI and PstI restriction sites ( Figure 1). The gene insert ran on the agarose gel at a position consistent with a predicted length of 1437 bp, as given by database entries for DYC18_RS18060. DNA sequencing confirmed that the DYC18_RS18060 gene had been cloned without mutation and was inserted into pTTQ18 in the correct orientation.
coli cells for expression studies. Recombinant DYC18_RS18060-His6 expression was tested from 50-mL cultures of cells that were grown in LB medium and induced with IPTG. Both SDS-PAGE and western blotting detected an amplified protein band with an apparent size of ~37 kDa in membrane preparations from induced cultures ( Figure 2).

Bioinformatic Analysis of DYC18_RS18060
Several databases and computational methods were used to obtain and analyse chemical and physical properties, predicted structure and function characteristics, and evolutionary relationships of the DYC18_RS18060 protein.
When the sequence of DYC18_RS18060 was aligned with those of experimentally characterised NCS-1 transporters, it shared only 25.0%, 24.4%, and 22.6% overall sequence identities with CodB (cytosine), PucI (allantoin) and Mhp1 (hydantoins), respectively ( Figure   5). From the sequence alignment between DYC18_RS18060 and crystographically defined Mhp1, four out of the nine residues involved in substrate interactions in Mhp1 are identical at the corresponding positions in DYC18_RS18060 and a further two are similar (Table 1).
Because the natural expression levels of membrane proteins are usually too low, amplified expression must be achieved [29,57]. Here we have demonstrated successful amplified expression of the R. erythropolis protein DYC18_RS18060 with a C-terminal His6-tag, as observed by an amplified band at ~37 kDa by SDS-PAGE and western blotting. Whilst the predicted molecular weight of DYC18_RS18060-His6 is 51.1 kDa, it is well known that membrane proteins migrate anomalously by SDS-PAGE at lower molecular weight positions than their actual [58]. Applying the correction factor for faster migrating proteins to the observed molecular weight (divide by 0.82) gives a corrected apparent molecular weight of 45.1 kDa, which reduces the error from 27.6% to 11.7%. Culture volumes can now be further scaled up to produce sufficient material for purifying up to milligram quantities of DYC18_RS18060-His6.
In the computational analysis of DYC18_RS18060, the high aliphatic index and GRAVY values reflect the high contents of aliphatic residues in the protein (11.5% alanine,  [27] shares 75.2% sequence identity with CodB and at the Mhp1-defined substrate binding positions, all nine residues are identical in VPA1242 and CodB. Overall, there is not strong evidence for DYC18_RS18060 having the same substrate specificity as any of CodB (cytosine), PucI (allantoin) or Mhp1 (hydantoins), so transport measurements using radiolabelled potential substrates [7,12,15] need to be performed for defining the substrate specificity of DYC18_RS18060. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins.

Conclusion
In order to obtain further information about the structure, function and evolutionary relationships of bacterial NCS-1 family transport proteins, we have overcome the challenge of cloning the R. erythropolis gene DYC18_RS18060 with introduction of a His6-tag and amplifying expression of the translated protein in E. coli inner membranes. Large-scale flask or fermentor cultures can next be used to produce sufficient quantities of membrane preparations to purify and reconstitute the DYC18_RS18060-His6 protein and to assess its purity, yield and thermal stability. The protein can then be analysed using a multitude of chemical, biochemical and biophysical techniques. Bioinformatics analysis of DYC18_RS18060 was consistent with the protein having an overall structural organisation of an NCS-1 protein, but its predicted role as a cytosine permease currently given in databases was not certain based on comparisons of residues involved in substrate binding in experimentally characterised bacterial NCS-1 proteins. The substrate specificity of DYC18_RS18060 will need to be determined by transport measurements using radiolabelled potential substrates. This work has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins.

Conflict of interest
The authors declare no conflict of interest.      The sequence of the DYC18_RS18060 protein was subjected to a BLAST search against proteins in the UniProt database (https://www.uniprot.org/blast/). The sequences of the top 250 results were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) [43], the nearest-neighbour phylogenetic results were extracted in Newick format and displayed as an unrooted phylogenetic tree using iTOL (http://itol.embl.de/index.shtml) [44].
The DYC18_RS18060 protein is indicated (red arrow). Some of the proteins are grouped (red ellipses) and some details are given about the host bacterial species and the putative function of the proteins as listed in the UniProt database.