Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies

2013 ◽  
Vol 34 (18) ◽  
pp. 2754-2759 ◽  
Author(s):  
Carlos E. Espinosa-de la Garza ◽  
Francisco C. Perdomo-Abúndez ◽  
Víctor R. Campos-García ◽  
Néstor O. Pérez ◽  
Luis F. Flores-Ortiz ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Purity Determination ◽  
Capillary Gel Electrophoresis

scholarly journals Nucleic Acids in Inclusion Bodies obtained from E. coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

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 Escherichia 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 are 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.

Separation and determination of some stereoisomers by capillary gel electrophoresis with cyclodextrin incorporated in polyacrylamide gel

1996 ◽  
Vol 354 (4) ◽  
pp. 451-454 ◽  
Author(s):  
Jin-Ming Lin ◽  
Tatsuro Nakagama ◽  
Hitoshi Okazawa ◽  
Xing-Zheng Wu ◽  
Toshiyuki Hobo
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Capillary Gel Electrophoresis

scholarly journals Nucleic Acids In Inclusion Bodies Obtained From E. Coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

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 Escherichia 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.

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