Modulation of recombinant protein solubility in ClearColi cells
In order to study the performance of APP fused to recombinant proteins in the endotoxin free ClearColi™ expression system, two model soluble proteins; iRFP (near-infrared fluorescent protein) and GFP (green fluorescence protein) were selected as scaffolds and fused to the surfactant-like peptide L6K2, previously described as APP (Fig. 1b) [13].
In transformed ClearColi cells, recombinant H6iRFP protein was equally distributed in both soluble and insoluble cell fractions (Fig. 1a). As expected, upon L6K2 fusion, a different distribution pattern was observed, where most of the protein was located in the insoluble cell fraction, suggesting an increased aggregation tendency for this fusion protein (Fig. 1a and 1b). The change in solubility pattern was achieved within 1 hour of induction and was maintained for up to 5 hours (Fig. 1b). As a model APP, with high ability to enhance aggregation tendency of recombinant proteins, VP1 from the capsid protein of the Foot-and-mouth disease virus [18, 19] was fused to GFP (Fig. 1b). As expected, most of the protein signal in the sample was detected in the insoluble cell fraction. In contrast, the recombinant H6GFPL6K2 was mostly detected in the soluble cell fraction. These results indicated that the aggregation propensity of a recombinant protein may be modulated by APP although the solubility tendency of the scaffold protein may counteract this effect (compare solubility of iRFP and GFP when fused to L6K2 in Fig. 1b). In the case of VP1, the aggregation tendency overcome the high solubility of the GFP, while GFP solubility was not affected by the L6K2 addition (compare solubility of GFP when fused to VP1 or L6K2 in Fig. 1b). At the same time, as the biopharmaceutical market expands, the need of recombinant proteins increases. For that reason, most of the factors affecting physical stability (aggregation) of peptides have been analyzed [23]. In addition, several methods based on algorithms are used for the prediction of the aggregation tendency of peptides and proteins [8, 24] in order to reduce or avoid formation of intermolecular interactions. However, since IBs have demonstrated the ability to maintain the original biological activity of the constituent protein, the study of this type of nanoclusters has gained interest for biopharmaceutical and industrial applications [25, 26].
Impact of APP length on protein solubility in ClearColi cells.
As the ability of L6K2 to reduce solubility of GFP was not significant while it was effective in iRFP, we decided to evaluate the effect of peptide length in the solubility of GFP. For that, we redesigned the H6GFPL6K2 recombinant gene to add at the C-terminus of the GFP sequence, different versions of the surfactant-like peptide L6K2 (Fig. 2a).The aggregating potential of L6K2 peptide was amplified by reiteration of leucine and lysine repeats in different positions (see Table 1) and analyzed by AGGRESCAN software [8]. Selected peptides displayed higher hot spot area (HSA) than the original L6K2 peptide. However, only L12K4 and L18K6 showed increased normalized hot spot area (NHSA) and increased average aggregation-propensity hot spot (a4vAHS).
Table 1
Predictions of “hot spots (HS)” of aggregation in aggregating polypeptides by AGGRESCAN [8]. HS: hot spot. HSA: hot spot area. NHSA: normalized HSA. a4vAHS: average aggregation-propensity in each HS.
Name | HS region | HS size | Sequence | HSA | NHSA | a4vAHS | Ref |
L6K2 | 1–6 | 6 | LLLLLLKK | 6.211 | 1.035 | 0.949 | [22] |
(L6K2)x2 | 1–14 | 14 | LLLLLLKKLLLLLLKK | 12.789 | 0.913 | 0.865 | This study |
(L6K2)x3 | 1–22 | 22 | LLLLLLKKLLLLLLKKLLLLLLKK | 19.367 | 0.880 | 0.842 | This study |
L12K4 | 1–13 | 13 | LLLLLLLLLLLLKKKK | 14.625 | 1.125 | 1.074 | This study |
L18K6 | 1–19 | 19 | LLLLLLLLLLLLLLLLLLKKKKKK | 23.025 | 1.212 | 1.171 | This study |
As previously observed, H6GFPL6K2 was detected in the soluble cell fraction of transformed E. coli cells (Fig. 2b) and consequently, the emitted fluorescence was homogenously distributed in the cytosol (Fig. 2c). The addition of the L6K2 derived peptides had a positive impact in protein aggregation tendency. As expected, the distribution of fluorescence in the transformed cells was detected in protein clusters (IB; Fig. 2c). In fact, we detected two different aggregation patterns in the L6K2 derived constructs. On the one hand, the proteins containing serial L6K2 repeats ((L6K2)x2 and (L6K2)x3) were preferentially detected in periplasmic areas around the cells, while the constructs containing longer Leucine/Lysine tracks (L12K4 and L18K6) were detected as fluorescent cellular pole aggregates. Therefore, the serial L6K2 repeats acted both as APP and periplasm localization signals since the fluorescence pattern revealed the clustering of signal in discrete aggregates on the periphery of the cell cytoplasm. In addition, the aggregation tendency in L6K2 repeats increased with the number of repeats while L12K4 presented an aggregation pattern like the observed in cells expressing the positive control VP1GFP. This aggregation tendency was not recorded in Western Blot analysis of the soluble and insoluble cell fractions (Fig. 2b), indicating that the aggregation tendency of the L6K2-derived peptides may be sensitive to the tested experimental conditions of protein extraction. This was not the case of the aggregation pattern of VP1GFP construct that was perfectly replicated under confocal laser scanning microscopy and SDS-PAGE (Compare VP1GFP data in Fig. 2b and 2c).
Antimicrobial activity of L6K2-containing recombinant proteins
During recombinant gene expression experiments of L6K2-containing constructs, the growth of transformed E. coli cells was compromised, especially in the case of L18K6 (data not shown). At that point, we reasoned whether the peptides were toxic to the cell by displaying antimicrobial activity. The modeling of the L6K2 derived peptides with PEP-FOLD 3 [27–29] displayed amphipathic alpha helices in all cases (Additional file 1: Figure S1). This configuration has been described in naturally produced or synthetic cationic antimicrobial peptides (AMP) which have been proposed as a potential new class of antimicrobial drugs (Huang 2010). The production of small peptides is difficult to be reached by recombinant technologies due to reduced stability, and alternative strategies have been taken to overcome such a main bottleneck [30]. One possibility is the fusion of AMP to partner proteins for a potential dual effect on the final product. First, the reduction of the toxicity of the AMP over the expressing host, and the improvement in the stability of the peptide in expression systems [31]. However, the study of their activity when fused to reporter proteins by genetic engineering has not been explored in depth. Examples of this strategy include the fusion between GWH1 [32] and GFPH6 [13, 33] and the secretory production of AMP-containing fusion partners [34]. In those studies, the fusion of the AMP to the N-terminus of recombinant protein preserved the bactericidal activity of the AMP even though with its C-terminus anchored by the fusion. Therefore, we analyzed the putative antimicrobial activity of the purified soluble versions of H6GFP-L6K2 and H6GFP-(L6K2)X2 proteins and compared these data with that obtained for purified GWH1-GFPH6. The results indicated that the antimicrobial activity of L6K2-containing recombinant proteins is strain specific (Fig. 3), being comparable to the antimicrobial activity of GWH1 peptide fused to GFP in E. coli cultures (Fig. 3b). In addition, the position of the peptide at each end of the scaffold protein did not appear to be relevant to the antimicrobial activity. On the other hand, the incubation of S. aureus with the proteins containing amphipathic alpha-helices had only a slight effect on cell viability under the tested conditions (Fig. 3a). Interestingly, the antimicrobial activity of the recombinant proteins was completely different when Micrococcus luteus cells were challenged. The addition of the purified proteins had a positive effect on cell viability at lower concentrations while at the highest protein concentration (8 µµολ/L) the cell viability dropped drastically (Fig. 3c).
As observed in Fig. 3b, the antimicrobial activity of the L6K2-containing constructs was detected in E. coli cultures at low protein concentrations. This mechanism may explain the cell growth inhibition observed in ClearColi cultures transformed with expression vectors with cloned L6K2-derived genes.
Pull-down effect on aggregation tendency of H6GFPL6K2
Aggregation of different proteins may be enhanced by the stereospecific interaction of APP in bacteria [18]. In that context, we reasoned that the aggregation ability of a recombinant protein with the same APP may enhance the aggregation tendency of H6GFPL6K2 when produced simultaneously in cells. For that purpose, we generated a dual expression vector including the gene encoding for H6iRFPL6K2, which displayed a high tendency to aggregate beside the gene coding for H6GFPL6K2 to be simultaneously expressed. In cells expressing at the same time the aggregation prone H6iRFPL6K2 construct and the soluble H6GFPL6K2 construct, the fluorescence of the GFP shifted from the cytoplasm to polar protein aggregates (IBs) (Fig. 4). The green fluorescence distribution in expressing cells was similar to the pattern observed when co-expressing VP1GFPH6 and H6iRFPL6K2.
The change in the aggregation propensity of the H6GFPL6K2 seemed to be directed by the pull-down ability of the L6K2 peptide present in the H6iRFPL6K2 construct. The intermolecular interactions between L6K2 present in the two proteins enhances the aggregation tendency of GFP. In the expressing cells, the newly formed H6GFPL6K2, when interacting with H6iRFPL6K2 with a high tendency to aggregate was dragged to the insoluble cell fraction. Therefore, it may be hypothesized that when two different proteins share aggregation prone domains, even if one of the proteins is still soluble, the protein with the highly aggregation propensity may lead the accompanying soluble protein to the insoluble cell fraction through coexpression. However, although the secondary structure of the iRFP and GFP proteins is not similar (Additional file 2: Figure S2), the effect of the iRFP scaffold protein in the aggregation enhancement of H6GFPL6K2 may not be ruled out. For that reason, a spectral variant of GFP (EBFP2; highly similar in amino acid sequence and secondary structure) was fused to VP1 domain generating VP1EBFP2H6 construct and coexpressed with GFP containing L6K2 (Fig. 5 and Additional file 3: Figure S3).
The distribution of the GFP fluorescence in cells simultaneously transformed with plasmids coding H6GFPL6K2 and VP1EBFP2H6 was homogeneously distributed in the cytoplasm of the cells, in agreement with the data obtained in the expression experiment of H6GFPL6K2 alone (compare the distribution of GFP fluorescence in Fig. 2c and Fig. 5a). On the other hand, the fluorescence emitted by EBFP fused to VP1 in those cells was mainly detected in polar IB as expected. When VP1GFPH6 was expressed along with VP1EBFP2H6, the GFP fluorescence was located exclusively at the poles of the cells, as IBs (Fig. 5b). The colocalization analysis of the fluorescence emission from both proteins indicated the preference of H6GFPL6K2 to aggregate in the presence of the same APP (Fig. 5c) ruling out an aggregating role of the scaffold protein in this process. Therefore, this result has a direct application for biopharmaceutical and biotechnological applications through protein engineering. The fusion of common APP to different therapeutic recombinant proteins can induce the co-localization of these two recombinant proteins in IBs, obtaining protein formulations with potential synergic activities.