Title: insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides

Abstract

Background: Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregation-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions.

Results: In this work we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP).

Conclusions: These data suggest that the molecular bases of protein aggregation are related to the net balance of protein aggregation potential and not only to the presence of APPs. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

Keywords: Antimicrobial peptides; Inclusion body formation; Intermolecular interaction; Protein aggregation; Recombinant protein.

Fig. 1

Effect of the addition of L6K2 in the aggregation propensity of fluorescent proteins. a Schematic representation of the recombinant genes is shown displaying protein domains by separate squares. H6 indicates the presence of a Hisx6 tag. PT corresponds to a linker with the indicated amino acid sequence. b Comparative aggregation propensity of iRFP and GFP in the presence of L6K2. VP1GFPH6 corresponds to a GFP fused to VP1 considered as positive control of the experiment. c Relative solubility (%) of recombinant proteins detailed in panel a analyzed by Western Blotting. Equivalent number of transformed ClearColi cells were lysed and soluble and insoluble cells fractions were separated. All proteins were detected with anti-his antibody

Fig. 2

Production of GFP-containing recombinant proteins fused to aggregation prone peptides in ClearColi. a Schematic representation of L6K2-containing GFP constructs. H6 indicates the presence of a Hisx6 tag. PT corresponds to a linker with the indicated amino acid sequence. VP1 corresponds to the VP1 structural protein of Foot-and-mouth disease virus with high tendency to aggregate. b Detection of GFP in soluble and insoluble cell fractions of ClearColi transformed with expression plasmids containing the corresponding recombinant genes. c Relative solubility (%) of recombinant proteins detailed in panel a analized by Western Blotting. d Confocal analysis of ClearColi cultures producing GFP with several aggregation-prone peptides. Arrows indicate the distribution of protein aggregates in the expressing cells

Fig. 3

Survival curves of microbial cells in the presence of putative antimicrobial peptides fused to GFP protein. a Staphylococcus aureus, b Escherichia coli and c. Micrococcus luteus. The results are presented as mean of two replicas for each analyzed point with corresponding standard error bar. Similar CFU where seeded on 96-well plates and the indicated protein concentrations were added to evaluate its effect on cell metabolism

Fig. 4

Confocal microscopy images of recombinant GFP in co-expression experiments. a Detection of H6GFPL6K2 in expressing H6iRFPL6K2 cells. b Detection of VP1GFPH6 in expressing H6iRFPL6K2 cells. A schematic representation of the corresponding constructs is depicted beside the confocal Microscopy images

Fig. 5

Overlay fluorescence images of H6GFPL6K2 and VP1GFPH6 with VP1EBFP2H6. a Detection of fluorescence emission from H6GFPL6K2 and VP1EBFP2H6 coexpressing cell cultures. b Detection of fluorescence emission from VP1GFPL6H6 and VP1EBFP2H6 coexpressing cell cultures. c Quantitative colocalization analysis of EBFP2 and GFP fluorescence signals in coexpression experiments. Overlap coefficients between the different fluorescences emitted by the fusion proteins VP1EBFP2H6/VP1GFPH6 and VP1EBFP2H6/H6GFPL6K2 expressed in ClearColi cells. Analysis performed from images obtained by confocal microscopy. *p < 0.001, one-way analysis of variance (ANOVA)