Abstract
BackgroundInclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model.ResultsIBs were isolated from E. coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA.ConclusionsThe results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.
Highlights
Escherichia 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 Krachmarova et al Microb Cell Fact (2020) 19:139 numerous tools for genetic manipulation [1 and references therein]
Inclusion bodies (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]
Purification of Human interferon-gamma (hIFNγ) IBs To study the type of nucleic acids co-aggregating with recombinant hIFNγ in E. coli cells we developed a three step procedure for purification of IBs from intact bacterial cells and subcellular components (Fig. 1)
Summary
Escherichia 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 Krachmarova et al Microb Cell Fact (2020) 19:139 numerous tools for genetic manipulation [1 and references therein]. At present, increasing evidence show that IBs have amyloidlike 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]. 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]. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model
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