Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli

Kim, Chang-Kyu; Choi, Jun-Ha; Lee, Seungbae; Lee, Sang-Mahn; Oh, Jae-Wook

doi:10.1007/s12010-013-0708-y

Cited by 8 publications

(3 citation statements)

References 25 publications

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“…Nowadays, several bioprocesses accumulate high percentage of recombinant protein in IBs to facilitate largescale recovery by centrifugation [53][54][55]. Hence, it is important to understand how culture conditions modify the aggregation properties inside IBs and how they maintain certain characteristics to extract active proteins or proteins with determined conformations.…”

Section: Introductionmentioning

confidence: 99%

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

et al. 2014

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Background: Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies.

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Section: Introductionmentioning

confidence: 99%

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

et al. 2014

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“…Several studies have earlier utilized different methodologies for improving the yield of G-CSF protein. Most of the studies have focused on modification of media composition, IPTG use (Vanz et al, 2008;Boubeva et al, 2012), ethanol utilization (Mishra et al, 2020), optimization of fermentation (Kim et al, 2014), or subsequent purification strategy/methodology (Wang et al, 2008;Li et al, 2012;Vemula et al, 2015) for improving the yields of G-CSF protein. Codon optimization of G-CSF for improved expression has been extensively used for expression in yeast (Maity et al, 2016) and E. coli (Gomes et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Optimizing Granulocyte Colony-Stimulating Factor Transcript for Enhanced Expression in Escherichia coli

Datta

2021

Front. Bioeng. Biotechnol.

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The human granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor used to prevent and treat neutropenia. G-CSF stimulates the bone marrow to produce infection-fighting granulocytes. Food and Drug Administration of the United States approved G-CSF in 1991 and its PEGylated version in 2002 as a prophylactic and therapeutic measure against neutropenia. Recombinant human G-CSF is produced in surrogate host Escherichia coli and is PEGylated at N-terminal. Besides neutropenia, G-CSF is also used in bone marrow transplantation for the mobilization and maturation of peripheral blood stem cells. Considering the requirement of producing G-CSF therapeutic in large quantities, construct designing for high expression is critical for the biopharmaceutical and industrial application. Earlier studies have employed approaches such as codon optimization, use of strong promoters, employment of protein tags, secretion signals, optimization of protein folding, etc., for increasing expression and yield of therapeutic proteins. In this study, it was observed that mRNA transcribed from the native human cDNA of G-CSF and the codon-optimized variant leads to low protein expression in E. coli. To understand the underlying reasons, the mRNA secondary structure of the 5′ end of the G-CSF transcript was analyzed. This analysis revealed the presence of stable secondary structures at the 5′ end of the G-CSF transcript, arising from the native human gene and even from the codon-optimized sequence. These secondary structures were disrupted through translationally silent mutations within the first 24 nucleotides of the transcript without affecting the protein sequence. Interestingly, through this approach, the G-CSF protein expression was increased 60 folds as compared to native G-CSF construct. We believe that these findings create a roadmap for optimization of G-CSF transcript for enhanced expression in E. coli and could be employed to increase the expression of other therapeutic proteins.

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“…Over the past decade, a number of functional proteins have been produced successfully by recombinant DNA methods [1, 2]. The most commonly used host cell is Escherichia coli ( E. coli ) because of high yield and low cost [3].…”

Section: Introductionmentioning

confidence: 99%

High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo

Wang

Jin

et al. 2015

BMC Biotechnol

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BackgroundIn our previous study, a novel liver-targeting fusion interferon (IFN-CSP) combining IFN α2b with plasmodium region I peptide was successfully constructed. IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells. The aim of the present investigation was focused on how to produce high levels of recombinant IFN-CSP and its in vivo anti-hepatitis B virus (HBV) activity.MethodsA modified DNA fragment encoding IFN-CSP was synthesized according to Escherichia coli (E. coli) preferred codon usage and transformed into E. coli BL21 (DE3) for protein expression. The induction conditions were systematically examined by combining one-factor experiments with an orthogonal test (L(9)(3)(4)). The antigenicity of the purified protein was characterized by western blot analysis. The in vivo tissue distribution were assayed and compared with native IFN α2b. HBV-transgenic mice were used as in vivo model to evaluate the anti-HBV effect of the recombinant IFN-CSP.ResultsThe results showed that the E. coli expression system was very efficient to produce target protein.ConclusionOur current research demonstrates for the first time that IFN-CSP gene can be expressed at high levels in E. coli through codon and expression conditions optimization. The purified recombinant IFN-CSP showed liver-targeting potentiality and anti-HBV activity in vivo. The present study further supported the application of IFN-CSP in liver-targeting anti-HBV medicines.

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Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli

Cited by 8 publications

References 25 publications

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

Optimizing Granulocyte Colony-Stimulating Factor Transcript for Enhanced Expression in Escherichia coli

High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo

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