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...
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doaj-9fa17f389db04130a924c405edb7de012020-11-25T02:41:22ZengBMCMicrobial Cell Factories1475-28592020-07-011911910.1186/s12934-020-01400-6Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gammaElena Krachmarova0Ivan Ivanov1Genoveva Nacheva2Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of SciencesInstitute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of SciencesInstitute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of SciencesAbstract 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 32P-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.http://link.springer.com/article/10.1186/s12934-020-01400-6Inclusion bodiesNucleic acidsE. coliRecombinant protein productionHuman interferon-gammaProtein aggregation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Elena Krachmarova Ivan Ivanov Genoveva Nacheva |
spellingShingle |
Elena Krachmarova Ivan Ivanov Genoveva Nacheva Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma Microbial Cell Factories Inclusion bodies Nucleic acids E. coli Recombinant protein production Human interferon-gamma Protein aggregation |
author_facet |
Elena Krachmarova Ivan Ivanov Genoveva Nacheva |
author_sort |
Elena Krachmarova |
title |
Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma |
title_short |
Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma |
title_full |
Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma |
title_fullStr |
Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma |
title_full_unstemmed |
Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma |
title_sort |
nucleic acids in inclusion bodies obtained from e. coli cells expressing human interferon-gamma |
publisher |
BMC |
series |
Microbial Cell Factories |
issn |
1475-2859 |
publishDate |
2020-07-01 |
description |
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 32P-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. |
topic |
Inclusion bodies Nucleic acids E. coli Recombinant protein production Human interferon-gamma Protein aggregation |
url |
http://link.springer.com/article/10.1186/s12934-020-01400-6 |
work_keys_str_mv |
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