Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Abstract

Background: Inclusion 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. Results: IBs 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 32 P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA. Conclusions: The 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.

Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

“…E. coli IBs while largely composed of self-aggregated protein, also contain traces of other bio-macromolecules including nucleic acids ( Krachmarova et al, 2020 ), and/or phospholipids ( Valax and Georgiou, 1993 ). Within the biotechnological context, proteins in IBs predominantly consist of expressed foreign proteins ( Rinas and Bailey, 1992 ; Valax and Georgiou, 1993 ).…”

Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli

“…They contain a high proportion of target protein and at the same time fewer undesirable proteins [ 40 , 41 , 42 ]. The optimization of recombinant proteins expressed in this form is relatively rare, but may represent a promising strategy for various insoluble proteins produced by E. coli [ 35 , 43 , 44 , 45 , 46 ]. In our previous work [ 26 ], we were able to produce biologically active NA in E. coli cells, and now we bring our focus to optimizing the conditions for the production of recombinant enzymes in inclusion bodies in order to maximize NA production.…”

Controlling the Quality of Nanodrugs According to Their New Property—Radiothermal Emission

“…IFNs are a group of cytokines, which are glycoproteins that function as multifunctional broad-spectrum bioregulators [19,20]. Modern IFN medicines are genetically engineered by growing recombinant strains of E. coli bacteria with IFN genes, which are obtained from leukocytes or connective tissue-human fibroblasts, built into bacterial DNA [21,22]. Pharmacologically, there are two types of IFNs: type I includes groups α and β, with sub-groups α2b, β1a and β1b, and type II includes the group of IFNs-γ [23,24].…”