E. coli is one of the most preferable microbial factory for production of recombinant proteins for research, diagnostics and medical use because of its easy, fast and cheap cultivation, well investigated genetics and physiology as well as for the availability of numerous tools for genetic manipulation [1 and references therein]. It is well known that the overexpression of eukaryotic proteins in E. coli often causes formation of inclusion bodies (IBs) [2], containing mainly improperly folded proteins [3]. For a long time, it has been believed that the aggregated proteins are immunogenic and partly or completely devoid of biological activity [4]. At present, increasing evidence show that IBs have amyloid-like structure and comprise aggregated as well as native folded proteins with preserved biological activity [2]. This fact, together with the mechanical stability and high porosity of the IBs has defined them as unconventional functional materials with a wide spectrum of applications in biotechnology and biomedicine [5, 6, 7]. Recently, first report towards in vitro preparation of tailored and chemically defined IBs with potential for clinical application was published [8]. In spite of these first steps, the large-scale clinical application of IBs is still hindered by their undefined heterogeneous composition and the presence of hazardous contaminants from the bacterial cell, especially endotoxins [9]. Thus IBs remain a main source for production of pure biologically active recombinant proteins for medical purposes that can be isolated upon cell disruption, solubilisation, subsequent refolding and purification [10]. Furthermore, to turn IBs aggregation into greatest use, new combinatorial approaches at each step was proposed [11].
Protein folding and IBs formation is extensively discussed in a number of reviews [1, 12, 13, 14, 15]. For a long time, protein aggregation has been considered as a process driven by hydrophobic interactions between fully-denatured protein molecules, however, increasing evidence indicate that protein aggregates are composed of partially unfolded or misfolded proteins linked by unspecific hydrophobic interactions [14, 16, 17]. Therefore, aggregation seems to be a competitive reaction to folding, depending on specific folding behaviour and conditions [18]. It is influenced by various factors such as protein size, presence of specific hydrophobic compartments in the molecule, pI, protein abundance [19, 20], high local concentration of the polypeptide chains emerging from ribosomes [21], limited amount of bacterial chaperones and proteases, which affect either folding or degradation of the unfolded or misfolded polypeptides [22, 23].
The chemical composition of IBs still remains obscure. It varies in a broad range and depends on the properties of the specific recombinant protein, fermentation conditions, host genetic background, IBs purification procedures, etc. [18]. Apparently, the major component of the bacterial IBs is the target recombinant protein [24, 25, 26, 27]. In addition, they can also contain various contaminants such as lipids, nucleic acids, endogenous cell proteins, chaperones, etc. [28]. Since chaperons assist in protein folding, they are the main cell components “controlling” protein aggregation [29]. Among them, the heat shock proteins IbpA and IbpB [30, 31, 32] and DnaK and GroEL [32, 33] have been found in bacterial IBs. There is evidence that the IBs contain also plasmid DNA [34], ribosomal RNA, RNA polymerase subunits [35, 36], ribosomal proteins L13 [36], L7 and L12 [24], elongation factor Tu [37], membrane proteins OmpF, OmpC, and OmpA [24], membrane phospholipids and cellular RNAs [38]. Based on these findings some authors assume that the protein aggregation in vivo occurs simultaneously with the protein synthesis [35, 36]. Taking into account that in most of these studies the IBs have not been precisely purified, Rinas & Bailey [24] suggest that the cellular ingredients found in IBs co-precipitate during their isolation.
The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. It has been shown that fully denatured mammalian proteins show unusually high solubility in nucleic acid-free pure water [39]. Since the recombinant proteins that are produced as pharmaceuticals are mainly mammalian proteins, the presence of nucleic acids in their preparation is critical for their refolding because the nucleic acids actively participate in the protein aggregation process via direct electrostatic interactions with partially folded or unfolded proteins. The literature survey shows that most of the studies on E. coli IBs carried so far are focused on protein composition and mechanisms of aggregation, whereas the data concerning nucleic acids are scarce and vague. Bearing in mind that the recombinant proteins manufactured for medical applications should be free of nucleic acids, we have focussed in this study on the content and nature of nucleic acids in highly purified E. coli IBs. As a model in this study we use IBs isolated from E. coli LE392 overexpressing human interferon-gamma (hIFNγ).