Purification and refolding of recombinant human interferon-gamma in urea–ammonium chloride solution

Petrov, S. V.; Nacheva, Genoveva; Ivanov, Iván

doi:10.1016/j.pep.2010.03.026

Cited by 21 publications

(24 citation statements)

References 25 publications

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“…As a result, the aggregates will be eluted faster than folded or partially folded proteins. If the mobile phase includes urea, a mild environment is formed in which proteins can refold during the elution process to form a more compact global structure (Fan et al ., ; Petrov et al ., ). The refolded proteins will then be eluted after they enter the pores in the gel particles.…”

Section: Resultsmentioning

confidence: 97%

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Yuan

Zhou

Yang

et al. 2014

Biomedical Chromatography

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Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

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Section: Resultsmentioning

confidence: 97%

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Yuan

Zhou

Yang

et al. 2014

Biomedical Chromatography

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“…Fig. 2 illustrates the signi cant difference in the protein composition of the IBs prepared by the standard procedure [41] and after the additional puri cation.…”

Section: Puri Cation Of Hifnγ Ibsmentioning

confidence: 99%

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

Krachmarova

Ivanov

Nacheva³

2020

Preprint

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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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“…Chromatographic refolding has also been conducted using adsorptive chromatography on various matrices such as ionexchange chromatography (IEC) [16][17][18] or hydrophobic interaction chromatography (HIC) media [19][20][21][22][23]. However, the denatured proteins often tend to aggregate in the course of adsorption-desorption cycle, which can impair yield of the operation [2].…”

Section: Introductionmentioning

confidence: 99%

Predictions of matrix‐assisted refolding of α‐lactalbumin: Process efficiency versus batch dilution method

Ryś

Pia̧tkowski

Antos

2014

Engineering in Life Sciences

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Predictions of matrix-assisted refolding of α-lactalbumin: Process efficiency versus batch dilution methodProtein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial-anderror method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to optimize the operating conditions. In this article, we suggest a design procedure for matrix-assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion-exchange chromatography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α-lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix-assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used.

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Purification and refolding of recombinant human interferon-gamma in urea–ammonium chloride solution

Cited by 21 publications

References 25 publications

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

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

Predictions of matrix‐assisted refolding of α‐lactalbumin: Process efficiency versus batch dilution method

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