Table 1.
Primers used for amplification of NS4A (1–48).
Figure 1.
ClustalW alignment [40] of the NS4A(1–48) coding sequence as found in the viral genome of DENV type 2 and following optimization for E. coli expression [21].
Figure 2.
Comparative expression of NS4A(1–48) constructs containing single fusion tags.
(A) SDS-PAGE analysis of the relative expression levels of NS4A fusions with ubiquitin (Ubi-NS4A(1–48)), glutathion-S-transferase (GST-NS4A(1–48)) and immunoglobulin-binding domain of streptococcal protein G (GB1-NS4A(1–48)). Aliquots of the expression cultures taken before (0) or 3 hours after IPTG induction (I) were applied. Aliquots of the supernatants after cell lysis (S) are shown as well. (B) TEV cleavage of the purified GB1-NS4A(1–48) fusion protein. Purified GB1-NS4A(1–48) fusion protein after size exclusion chromatography before (−) and after (+) TEV digestion together with a molecular weight marker (M; M3546, Sigma) were applied.
Table 2.
Average yields and purities of the studied NS4A(1–48) fusion proteins and of the resulting NS4A(1–48) target peptides obtained from 1 L of culture.
Figure 3.
Expression/cloning region of pGEX-GB1-NS4A(1–48).
The GB1-NS4A(1–48) sequence was inserted into pGEX-4T-2 between the EcoRI and XhoI sites. Protease recognition motifs are underlined while the cleavage sites are marked by triangles. The two thrombin sites, which originate from the vector backbone of pGEX and pGEV, respectively, are shown in brackets and were not used in our protocol. Note that TEV digestion produces a native NS4A peptide.
Figure 4.
Expression of NS4A(1–48) wild type and mutant peptides using a dual GST-GB1 tag.
(A) Relative expression levels of GST-GB1-NS4A(1–48) and the GST-GB1-NS4A(1–48; L6E, M10E) mutant were analyzed by SDS-PAGE using aliquots of the expression cultures. Shown are samples obtained from culture at 0 (0) or 3 hours (I) following IPTG induction or the supernatant after cell lysis (S). (B) TEV digest of GST-GB1-NS4A(1–48) wild type and mutant protein fusions. Aliquots of GSH-purified supernatants of wild type and mutant fusion proteins before and after TEV cleavage are shown. Note that besides the GST-GB1-NS4A(1–48) full-length product also shorter fragments, likely GST-GB1 and other truncation fragments, marked by asterisks were produced, which are present in the GSH-purified samples already prior to TEV cleavage. Because staining of free NS4A(1–48) peptides is very faint under the conditions used, the progress of the TEV digest is monitored by observing the decrease of the band for the dual tagged GST-GB1-NS4A(1-48) fusion protein in parallel with an increase of the band for the free GST-GB1 dual tag. A densitometric analysis of the respective bands revealed a cleavage efficiency of approximately 50% for the wild type peptide.
Figure 5.
Optimization of TEV protease cleavage conditions.
(A) A schematic model explaining the resistance of the GST-GB1 dual tagged wild type NS4A(1–48) peptide to TEV cleavage. Scissors illustrate the protease while the triangles represent the position of the cleavage sites. GST induced oligomerization of wild type NS4A(1 48) may block the protease cleavage site. (B) Analysis of the urea tolerance of TEV protease activity. GST-GB1 tag removal from NS4A(1 48; L6E, M10E) in the presence of different concentrations of urea (M). Fusion peptides were incubated with TEV protease at 20°C for 16 h at a fusion peptide to protease molar ratio of approximately 100. (C) Binding properties of GST-GB1-NS4A(1–48) to GSH sepharose in the presence of urea. Equal amounts of the fusion peptide were subjected to a mini-scale GSH-affinity chromatography in the presence of different concentrations of urea (M). Input, flow-through and elution fractions were analyzed by 15% SDS-PAGE. (D) TEV cleavage efficiency of wild type NS4A(1–48) peptide in the presence of urea. GST-GB1 tag removal from NS4A(1–48) in the presence of different concentrations of urea given in Molars. Fusion peptides were incubated in the absence (−) or presence (+) of TEV protease at 20°C for 16 h. The different “+” font sizes indicate the increasing amounts of TEV protease with fusion peptide to protease molar ratios of approximately 100, 50 and 10. The progress of the TEV digest is monitored by observing the decrease of the band of the dual tagged GST-GB1-NS4A(1–48) fusion protein and a parallel increase of the free GST-GB1 dual tag band (B, D).
Figure 6.
Purification of recombinant NS4A(1–48) wild type and mutant peptides.
The 15% SDS-PAGE gel containing samples from various steps in the purification procedure is shown on the left. The wild type peptide is shown in (A) while the mutant is shown in (B). The supernatant after cell lysis is shown in lane 1. The lysate containing the peptide was loaded on a GSH sepharose column (lane 2), and cleaved by TEV protease on the column, the cleaved protein is shown in lane 3. The GSTfusion tag remained bound to the column, while the NS4A(1–48) peptide was collected from the flow-through (lane 4). The peptide was further purified by size exclusion chromatography (lane 5). The mutant peptide was purified using the same strategy (B). The respective size exclusion chromatography profiles (HiLoad 16/60 Superdex 75 prep grade) of the flow-through fraction from the TEV protease on column cleavage step - mainly containing TEV protease and NS4A(1–48; L6E, M10E) or NS4A(1–48) peptides - are shown on the right with the matching SDS-PAGE analysis of the NS4A containing fractions.
Figure 7.
Recorded 2D (1H, 15N)-BEST-TROSY spectra of the purified NS4A peptides.
Spectrum of 0.5[15N]-NS4A(1–48) wild type (A) and of 1 mM mutant (L6E, M10E) peptide (B) in 50 mM sodium phosphate buffer, pH 6.8. Data were recorded at 30°C.