Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli

Objectives To purify and characterize the glutathione binding protein GsiB of glutathione importer (GSI) in Escherichia coli (E. coli). Results The coding sequence of GsiB was cloned from E. coli MG1655 and expressed in BL21(DE3). GsiB protein was expressed and purified to homogeneity using Ni-affinity and gel filtration chromatography. SDS-PAGE of purified GsiB showed a single protein band of molecular mass 56 kDa, while native gel showed two bands around 56 kDa and 110 kDa. Gene knockout showed that GsiB was essential for GSI mediated glutathione import. Interactions of GsiA, B, C, and D were determined using bacterial two-hybrid method. Without glutathione, GsiB showed no direct interaction with the other three proteins. However, GsiB could interact with GsiC and GsiD when using glutathione as sole sulfur source. Conclusions GsiB functions in E. coli was characterized which could help elucidate the glutathione import mechanism in gram-negative bacteria.


Introduction
Glutathione ( -L-glutamyl-L-cysteinyl-glycine; GSH) is the most important endogenous antioxidant across all kingdoms of life. GSH is a tripeptide consisting of glutamate, cysteine, and glycine amino acids. GSH is distributed ubiquitously and usually attains mM concentrations in human body. It plays an important role in maintaining the intracellular redox homeostasis [1], as well as in the detoxification of xenobiotics and their metabolites [2,3]. GSH also functions in salvage of cysteine [4] and cell signaling [5,6]. In cell, glutathione manifests predominantly in thiol-reduced form (GSH) [7]. A small quantity of glutathione is in oxidation form, such as glutathione disulfide (GSSG) or disulfides with target proteins [8]. The intracellular GSH homeostasis can be maintained by different pathways, including de-novo synthesis, GSH redox cycling, and direct uptake [9]. GSH import by bacteria may serve as organic sulfur resource [10][11][12]. However, the mechanisms that underpin glutathione uptake still need further investigation.
The specific glutathione importer (GSI) in bacteria was identified in 2005 [10]. This importer consists of GsiA, -B, -C, and-D, which encodes ATP binding protein, glutathione binding protein, and two inner-membrane components [13]. The specific glutathione recognition is mediated by GsiB and import is ATP dependent [10,14].
Herein, the spectrum of expression, purification and characterization of GsiB from E. coli was described. The in vitro and in vivo functions of GisB were investigated. Studies of GsiB will help to clarify the mechanism of specific glutathione import in bacteria.

Materials and Methods
. . Strains, Plasmids and Chemicals. The E. coli strains MG1655, BL21 (DE3), DH5 , and plasmid plou3 were preserved in our laboratory. Vectors pET11a-link-NGFP, pMRBAD-link-CGFP, pN-Z, and pC-Z were gifts presented by Professor Lynne Regan of Yale University. Pfu polymerase and T4 DNA ligase and restriction enzymes were purchased from NEB. Other chemicals were purchased from Sangon Biotech.
. . Heterologous Expression of GisB Protein. GsiB gene (accession number: HM217135) was amplified from genomic DNA of E. coli MG1655 using primers GsiB-F and GsiB-R ( Table 1). The gene was cloned into expression plasmid plou3 and transformed into BL21 (DE3). MBP (Maltose binding protein) was used as fusion prtoein. BL21 (DE3) was grown in Luria-Bertani (LB) medium (containing ampicillin 100 g/ml) at 37 ∘ C till OD 600 reach about 0.5-0.6. Protein expression was induced by adding 0.1 mM IPTG and grown at 22 ∘ C for 20 h. Cells were harvested by centrifugation at 5000 g for 15 min at 4 ∘ C.
. . Purification of GsiB Protein. The cell pellet was resuspended in 50 mM Tris/HCl buffer (pH 7.5) containing 100 mM NaCl, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 M lysozyme lysozyme, and 1 M DNaseI. The cell was disrupted by using homogenizer FB-110X (LiTu, China) with 800 MPa. The sample was centrifuged at 8,000 g for 15 min and supernatant was loaded onto Ni 2+ affinity column (GE Healthcare). The protein was washed with 50 mM Tris/HCl pH 7.5, 300 mM NaCl, and 30 mM imidazole. GsiB-MBP was eluted with 50 mM Tris/HCl pH 7.5, 300 mM NaCl, and 300 mM imidazole. Imidazole was removed by desalting. The protein was then digested with TEV protease and MBP was removed by MBP column. GsiB was further purified using Superdex 200 column (GE Healthcare) (buffer contains 50 mM Tris/HCl pH 7.5, 300 mM NaCl, and 5 % (v/v) glycerol). The purity of GisB was determined by SDS-PAGE. Native gel was performed to analyze protein conformations. The protein was concentrated to 5 mg/ml, which was measured by Nanodrop 2000 (Thermo Scientific).
. . GsiB Interacts with Other Components. GFP fragments reassembly protocol was used to determine interaction of GsiB with other GSI components [16,17]. GsiA, GsiB, GsiC, and GsiD were cloned into pMRBAD-link-CGFP and pET11a-link-NGFP (With primers in Table 1). Any two recombinant plasmids carrying N-and C-fragment of GFP were simultaneously transformed into BL21(DE3) with 10 ng of DNA. The recombinant cells were plated onto selective agar medium, containing kanamycin (35 g/ml) and ampicillin (100 g/ml). Single colonies were selected and incubated with appropriate antibiotic. Fresh overnight culture was diluted (1:1000 (v/v)) and 100 l medium was plated onto screening medium, containing 0.2% arabinose and 10 M IPTG. The plates were incubated at 20 ∘ C for 2 days to induce protein expression and interacion.
The M9 medium plate [18] was also made with MgSO 4 replaced by MgCl 2 . Reduced glutathione (1 mM, ≥98%) was added as only sulfur source to characterize the interaction.
. . Characterization of Glutathione Binding Activity of GsiB. The glutathione binding activity of GsiB was determined by native gel. Purified GsiB was incubated with 5 mM GSH or GSSG and separated on 12% Native-PAGE. The native gel electrophoresis was performed basing on theoretical pI 8.22 of GsiB. The protein samples incubated with glutathione were also analyzed by 12% SDS-PAGE.
. . GsiB In-Vivo Function Assay. It was supposed that gramnegative bacteria uptake glutathione mainly throughglutamyltranspeptidase (GGT) pathway or GSI complex [10]. The GsiB and ggt gene of E. coli MG1655 were deleted with Red recombination system [19,20]. The kanamycin resistant DNA was amplified from pKD4 with primers GsiB del-F and GsiB del-R ( Table 1). The PCR product had 58 bp upstream and 58 bp downstream homologous to adjacent sequence of GsiB, which was digested with DpnI and gel-purified. pKD46 was transformed into E. coli MG1655 by CaCl 2 method. The cell with pKD46 was grown in SOB medium at 30 ∘ C to an OD 600 of around 0.5. 2 mM L-arabinose was added 1 h before cell collection. Competent cell was made by washing with 10% glycerol. 50 ng of PCR product was mixed with 50 l of competent cell. Electroporation was performed using MicroPulser (Bio-Rad) with a 0.1 cm chamber.
ggt gene was deleted as above. The chloramphenicol fragment was amplified with 60 bp upstream and 60 bp downstream homologous to adjacent regions of ggt (primer GGT del-F and GGT del-R) ( Table 1). GsiB and ggt gene deletion was verified by PCR with primers Del-F and GsiB-Del-R or GGT-Del-R ( Table 1).
The cell growth and glutathione uptake curves of mutant strains were measured. M9 minimal medium [18] was used with MgSO 4 replaced by MgCl 2 . Glutathione (1 mM, ≥98%) was served as the only sulfur source. GsiB was cloned into pBAD24 (primers GsiB-F and GsiB-R) and transformed into mutant strains to compensate for gene defection.

Results
. . Expression and Purification of GsiB. The GsiB gene was amplified from E. coli MG genome and cloned into plou3 vector which was derived from pMAL-c2X. A 6×His tag and a TEV protease cleavage site were added before and behind MBP to facilitate protein purification. The resultant plasmid was denominated plou3-gsiB (Figure 1(a)). The insertion of GsiB gene was confirmed by DNA sequencing.
GsiB was expressed in BL21(DE3) and expression condition was optimized. Induction with 0.1 mM IPTG at 22 ∘ C for 20 h will give high productivity of soluble GsiB (Figure 1(b)). GsiB-MBP fusion protein was firstly purified by Ni 2+ column and then digested with TEV protease. The MBP was removed by Ni 2+ and MBP column. The protein was further purified by gel filtration. 12% SDS-PAGE analysis showed that the molecular mass of GsiB was about 56 kDa with purity in excess of 90% (Figure 1(c)). The protein was concentrated to 5 mg/ml and used for glutathione binding activity assay. Approximately 0.8 mg of GsiB protein was obtained from per liter of LB medium.
Western blot confirmed the expression of GisB. As there was a 6×His tag at the N terminal of GsiB-MBP, anti-6×His antibody was used here (Figure 1(d)).
. . Characterization of Glutathione Binding Activity of GsiB. The purified GsiB was shown to have two different conformations in native gel. GsiB protein might be present as monomer and dimer (Figure 2(a)). GsiB protein was incubated with reduced (GSH, ≥98%) and oxidized (GSSG, ≥98%) glutathione at 25 ∘ C for 2 h. However, after incubation with GSH or GSSG, there was no protein band that could be detected in native gel (Figure 2(a)). This phenomenon might be explained by GsiB conformational change, caused by binding GSH or GSSG. To confirm if the protein was degraded in Figure 2(a), SDS-PAGE was performed. The result showed that GsiB protein was not degraded (Figure 2(b)). The conformational change might confer change of surface charge of GsiB protein.
As reversing of cathode and anode position showed protein band in native gel (data not shown). However, the protein band run very slow in the gel. This might because of weak surface charge.
Incubation at 25 ∘ C would promote GsiB to form another band with molecular weight of about 110 kDa in SDS-PAGE (Figure 2(b)). In the meantime, binding GSH or GSSG would obviously reduce the top band ratio (Figure 2(b)). The top band might be dimer of GsiB, which was not separated by denature at 95 ∘ C for 3 min. To verify this conjecture, a 6×His tag was added at the C terminal of GsiB (primer GsiB-F and GsiB-6His-R) ( Table 1). Using anti-6×His antibody, western blot showed both the two bands were GsiB protein (Figure 2(c)).
The results indicated that GsiB could bind both GSH and GSSG. Binding with substrate would induce conformational change of GsiB.
The results showed that GsiB could not interact with the other three proteins on LB plate. Without binding glutathione, GsiB might present in inactive conformation.   It was speculated that glutathione binding might promote conformational change, which would facilitate GsiB to interact with other components. To verify this hypothesis, M9 medium plate with glutathione as sole sulfur source was made and the interaction was characterized. As shown in Figure 3, GsiB could interact with transmembrane proteins GsiC and GsiD. However, GsiB showed no interaction with GsiA. It might be associated with their different cell locations. GsiA and GsiB were predicted to be located in the cytoplasm and periplasm of cell, respectively [13].

. . GsiB Was Essential for GSI Mediated Glutathione Import.
The GsiB and ggt gene in E. coli were replaced by kanamycin and Chloramphenicol resistant gene. The gene deletion strains were named GsiB, ggt, and GsiB ggt. The deletion was verified by PCR.
The cell growth and glutathione uptake curves were measured, using M9 medium with glutathione as sole sulfur source. The results suggested that GsiB ggt strain grew much slower in glutathione containing M9 medium than in LB medium. The slow growth rate could be somewhat compensated by transformation of pBAD24-gsiB (Figure 4(a)).
ggt could uptake glutathione at a lower rate than wild type and GsiB. However, the glutathione import in GsiB ggt strain was undetectable (Figure 4(b)). The results depicted that GGT pathway was more effective, which might mediate more glutathione uptake than GSI. As GsiB gene deletion could block GSI mediated glutathione uptake, which was compensated by pBAD24-gsiB (Figure 4(b)). GsiB was essential for GSI mediated glutathione uptake.

Discussion
Glutathione is the most abundant small molecular weight thiol containing antioxidant in living cells and plays a plethora of cellular roles. GsiB is the glutathione binding protein of GSI, which specifies the transporter. Putting deep insights into functions of GsiB will help to elucidate the mechanism of specific glutathione import.
The Ni 2+ -NTA column could enrich His-tagged GsiB, comprising more than 90% of total proteins. MBP was used as fusion protein which can assist protein folding. The MBP fusion can be removed by MBP column. MBP used here promoted solubility of GsiB. By using different tags and purification columns, the purification of proteins could be efficient.
Lower inducing temperature and IPTG concentration would contribute to soluble expression of GsiB. Induced at 22 ∘ C with 0.1 mM IPTG, GsiB was expressed mainly in soluble fraction. Protein expression was confirmed by Western blot. High yield of pure GsiB protein will contribute to future biophysical and biochemical studies.
The freshly purified GsiB showed only one band in SDS-PAGE and two bands in native gel. GsiB might be present as monomer and dimer here. However, after incubation at 25 ∘ C, another protein band appeared in SDS-PAGE. This band might be dimer of GsiB, as the molecular weight was about 110 kDa. This top protein band was stable, which would not be separated by denature at 95 ∘ C. However, the top band could be reduced by incubation with GSH or GSSG. As shown by crystal structure (PDB ID: 1UQW), the N-terminal fragment was located at surface of GsiB. Although not included in the structure, Cys23 was speculated to locate at the surface of full length GsiB, which might form disulfide bond between proteins. The band could be disrupted by glutathione. Or the binding of GSH or GSSG could block Cys23 site, which probably affect disulfide bridge formation. In the meantime, crystal structure of GsiB was shown to have A and B chains. Chain A and B both contained a GsiB protein. The two chains of GsiB protein had different structures, which was in consistence with two conformations in native gel.
GsiB didn't interact with the other three proteins of GSI on LB plate. However, GsiB could interact with the innermembrane proteins GsiC and GsiD when glutathione was used as sole sulfur source. It was speculated that GSI had two state: 'open' and 'close' . The state of GSI depends mainly on GsiB protein conformation. Without binding glutathione, GsiB would present in 'inactive' conformation and will not interact with GsiC or GsiD. GSI would be at 'close' state. Binding with glutathione would change GsiB to 'active' conformation, which facilitate GsiB to interact with innermembrane channel. GsiA could hydrolyze ATP to support glutathion import and GSI would 'open' . The 'open' state of GSI required GsiB binding with glutathione and GsiA, B, C, D to interact with each other. In summary, binding with GSH or GSSG would change GsiB protein conformation from 'inactive' to 'active' . The activated GsiB interacted with GsiC and GsiD and substrate was then transferred into innermembrane channel. The transportation of GSH and GSSG was powered by GsiA hydrolyzing ATP. After glutathione import, GsiB was released from the complex and wait for another transportation. GisB was deleted in E. coli to determine the in-vivo function. The growth of GsiB was not affected when using glutathione as sole sulfur source. This is because the strain could uptake glutathione from the medium by GGT pathway. Figure 4(b) showed that GGT pathway could mediate much more glutathione import than GSI. Glutathione imported by GGT was then hydrolyzed to glutamic acid and cysteinylglycine [21]. Cysteinylglycine was cleaved into cysteine and glycine by aminopeptidases A, B, and N and dipeptidase D. So glutathione could serve as sulfur source for GsiB to survive and grow. However, the growth of GsiB ggt was affected with glutathione as sole sulfur source. As shown in Figure 4(b), the glutathione uptake by GsiB ggt strain was undetectable. The glutathione import was compensated by transformation of pBAD24-gsiB. The results showed that GsiB was essential for GSI mediated glutathione import.
Collectively, the glutathione binding protein GsiB from E. coli was expressed and characterized. Investigation of biological functions and protein interactions of GsiB would help to elucidate the specific glutathione import mechanism.

Data Availability
The glutathione import related data used to support the findings of this study are included within the article. The information of plasmids used in this study is available from the corresponding author upon request.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.