4. Metodología implementada
4.1. Generalidades
Plasmids encoding individual fusion partners, i.e. GFPuv, Eno, MBP, MreB and NusA were generated for expression in E. coli at 37 oC. As expected, these four bacterial proteins displayed greater than 90% solubility even when overexpressed to >20% of the total cellular protein. GFPuv also accumulated significant amount of soluble material of greater than ~50% of total expressed protein, which is in agreement with previous observations (Figure 7) (Fukuda et al., 2000).
Figure 7. Solubility of GFP and nonfused protein partners upon expression in E. coli. Protein samples were examined by SDS-PAGE and staining with Coomassie brilliant blue. Protein in the supernatant (S) and pellet (P) fractions is indicated. Marker proteins (M) are in descending order: 200 kDa, 150 kDa, 120 kDa, 100 kDa, 85 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa and 20 kDa.
In addition to selecting appropriate fusion partners, the properties of the amino acid sequences used as domain linkers should be considered when performing protein fusion studies. To test whether the linker between GFPuv and the fusion partner had an influence on the folding of the resulting fusions, we selected six different linkers which varied in their length, predicted conformational rigidity, hydrophobicity and net charge (Table 2). The resulting 24 GFPuv fusion proteins were generated with GFPuv as the C-terminal fusion partner of Eno, MBP, MreB and NusA (Figure 8).
Figure 8. Constructs for expression of GFP fusions in E. coli
Diagram of the different phage T7 promoter-driven constructs for expression of GFP fusions in bacteria, with each component represented by a box. Fusion partners and linkers are listed underneath each box. Linker characteristics are given in Table 2.
After expression in E. coli of these fusions to levels similar to those of the non-fused proteins, we observed that the solubility of the fusion proteins was dramatically lower than that of their non-fused counterparts in all cases tested. As shown in Figure 9A, >90% of the Eno-, MreB-, and NusA-GFPuv fusions and >80% of the MBP-GFPuv fusions were present in the insoluble fraction, regardless of which linker was used. Substantial aggregation was observed when the robustly folding proteins were present at the N-terminus of the fusion proteins, which suggests that the C-terminal GFP moiety interfered with the folding of the more N-terminal partner. This also indicates that the N-terminal fusion partners failed to acquire their correct structure by a sequential, co-translational mechanism while the GFP moiety was still emerging from the ribosome.
To be certain that indeed fusing the domains per se was responsible for the observed misfolding instead of, for example, a steric problem due to the incorrect fusion order, we generated constructs where GFPuv was used as the N-terminal fusion partner to the bacterial proteins. As has been repeatedly described, robustly folding “carrier proteins” such as NusA or MBP, are fused to the C-terminus of aggregation-prone eukaryotic proteins in an attempt to aid their solubilization. Therefore it remains possible that GFPuv, a eukaryotic moiety, could fold better if fused to carrier proteins at its N-terminus. Since there was no major difference among the linkers tested, we decided to utilize either L16 or L25 for further experiments. Additionally, since the linker sequence present in front of GFP in the fusions mentioned above could potentially be responsible for the observed misfolding, we decided to include in this analysis N-terminal GFPuv fusions containing a sequence identical to linker L5 as an N-terminal segment (Figure 8).
Interestingly, the resulting eight fusions displayed an even lower solubility when compared to their C-terminal GFPuv fusion counterparts (Figure 9B). This is particularly obvious for the GFPuv-MBP fusions, which are present almost exclusively in the insoluble fraction, in contrast to the MBP-GFPuv fusions, in which about 15-20% of the material was
soluble (compare Figures 9A and 9B). This suggested that the fusion event itself, probably by increasing the complexity of the folding reaction, was responsible for misfolding. In conclusion, our results showed that domain order and the presence of additional sequences at the extreme N-terminus of GFPuv are not critical for fusion protein misfolding.
Figure 9. GFP fusion proteins aggregate upon expression in E. coli
(A) Solubility of C-terminal GFP fusion proteins upon expression in E. coli examined by SDS-PAGE and staining with Coomassie brilliant blue. Protein in the supernatant (S) and pellet (P) fractions for each fusion protein resulting from the combination of the six linkers (listed on top) and the four fusion partners (listed on the left) is indicated. (B) Solubility of N-terminal GFP fusion proteins upon expression in E. coli examined as above. Protein in the supernatant (S) and pellet (P) fractions of each fusion protein resulting from the combination of the two linkers (listed on top) and two fusion partners (listed on the left) is indicated. Proteins with the original GFP N-terminus are indicated as N- and those with an additional L5 linker sequence as L-N-.
In order to assess the folding efficiency of GFPuv fusions in the eukaryotic cytosol, we analyzed these proteins by recombinant expression in S. cerevisiae (Figure 10).
Figure 10. Constructs for expression of GFP fusions in S. cerevisiae
Diagram of the different GAL1 promoter-driven constructs for expression of GFP fusions in yeast, with each component represented by a box. Fusion partners and linkers are listed underneath each box. Linker characteristics are given in Table 2.
Surprisingly, we observed that a majority of total protein was soluble in each case tested, regardless of the fusion position of GFPuv (Figure 11A and 11B). In vivo, cellular localization of green fluorescence supported the notion that both domains of the fusion proteins were correctly folded when expressed in yeast (Figure 11C). For instance, the transcription factor NusA is a nucleic acid binding protein and its GFPuv fusions displayed clear accumulation in the nucleus, most probably as a result of the interaction between NusA and yeast chromatin. Similarly, fusions of GFPuv with the filament-forming protein MreB were present in filamentous structures reminiscent of actin cables (Doyle and Botstein, 1996). On the other hand, enolase and MBP would be expected to remain soluble
in the yeast cytosol, and these proteins showed a diffuse cytosolic distribution when fused to GFPuv.
Figure 11. GFP fusion proteins are highly soluble upon expression in S. cerevisiae (A) Solubility of C-terminal GFP fusion proteins upon expression in S. cerevisiae examined by SDS-PAGE, followed by immunoblotting with anti-GFP monoclonal antibodies. Protein in the supernatant (S) and pellet (P) fractions of each fusion protein resulting from the combination of the six linkers (listed on top) and the four fusion partners (listed on the left) is indicated. (B) Solubility of N-terminal GFP fusion proteins upon expression in S. cerevisiae examined as above. Protein in the supernatant (S) and pellet (P) fractions of each fusion protein resulting from the combination of the two linkers (listed on top) and two fusion partners (listed on the left) is indicated. (C) Fluorescence microscopy of GFP fusion proteins in living S. cerevisiae cells. The indicated fusion proteins contained linker L16. Scale bars represent 10 µm.