Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation

Babaeipour, Valiollah; Abbas, Mahdi Pesaran Haji; Sahebnazar, Zahra; Alizadeh, Reza

doi:10.1007/s00449-009-0380-3

Cited by 9 publications

(10 citation statements)

References 19 publications

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“…Most commonly, induction is carried out during the exponential growth phase when cells are most actively dividing and when the protein expression machinery is believed to be the most active [18,21,22,36]. In contrast, our studies indicated that maximum SELP volumetric productivity is achieved by induction at the beginning of the stationary phase (8 hrs.…”

Section: Resultsmentioning

confidence: 66%

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

et al. 2013

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BackgroundSilk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.ResultsThis study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.ConclusionsWe have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.

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Section: Resultsmentioning

confidence: 66%

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

et al. 2013

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“…On the other hand, the production of the recombinant protein referred to as inclusion body has not only disadvantages, but also has some advantages. 15,18 The primary advantage is the ease of purification of IBs and it contains high degree purity of target protein.…”

Section: 14mentioning

confidence: 99%

Recombinant human G-CSF production as a protein based drug candidate for hematology and oncology

Tayhan¹

2019

International Journal of Chemistry and Technology

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The human granulocyte colony stimulating factor (hG-CSF) is a member of the CSF family. The first purpose of this study was production of recombinant human G-CSF (rhG-CSF) which is an important therapeutic protein for angiogenesis based clinical applicaitons. rhG-CSF was produced as inclusion body in the Escherichia coli strain BL21 (DE3) pLysE with pTOLT vector system. The rhG-CSF was purified by Ni-NTA agarose afinity chromatography and characterized with SDS-PAGE analysis. The effects of this therapeutic protein on cell viability was measured by MTT assay. Additionally, angiogenic potential of produced rhG-CSF was investigated via HUVEC cell line by in vitro scratch assay. As a result, the purified protein induced cell proliferation and the EC50 value of protein based drug candidate was 0.051 mM. Additionally, it was determined that the migration ability of the HUVECs was promoted by rhG-CSF in a concentration-dependent manner by in vitro scratch assay.

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“…Efficient cell lysis and maximum protein extraction yields are vital to high‐quality recombinant protein purification. Mechanical lysis by high‐pressure homogenization or sonication or lysis by freeze–thaw procedures with lysozyme is equivalent in most cases [18,19]. After the appropriate cell disruption, IBs are easily separated by high‐speed centrifugation.…”

Section: Introductionmentioning

confidence: 99%

“…After the appropriate cell disruption, IBs are easily separated by high‐speed centrifugation. Further purification of IBs can be achieved by washing them with detergents, low concentration of salt, and or urea as the best approach [18,20]. With proper isolation and washing processes, IBs with a purity of more than 95% can be obtained from E. coli [21].…”

Section: Introductionmentioning

confidence: 99%

Efficient process development for high‐level production, purification, formulation, and characterization of recombinant mecasermin in Escherichia coli

Mofid

Babaeipour

Jafari

et al. 2020

Biotech and App Biochem

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Overproduction of recombinant mecasermin was achieved by investigation of effect of three factors, temperature, inducer amount, and culture media, at three levels according to the Taguchi statistical design in Escherichia coli in a bench‐scale bioreactor. In optimal conditions (induction temperature 28 °C, terrific broth with glucose (TB+G) medium, with 0.1 mM IPTG as inducer) 0.84 g/L mecasermin with expression levels of 38% of total protein and 4.13 g/L final dry cell biomass was produced, that is one of the highest values of recombinant protein has been reported in the batch system. The cell disruption was done by lysozyme pretreatment with sonication to the efficient purification of mecasermin. The isolated and washed inclusion bodies were solubilized in Gdn‐HCl at pH 5.4 and folded with glutathione and purified with gel filtration. The purified rhIGF‐1 (mecasermin) was formulated with arginine. Mecasermin protein remained t stable at 4 °C for up to 2 years. The quantitative and qualitative control indicated that mecasermin is expressed correctly (without the initial methionine by mass spectrometry), pure (without endotoxin and other protein impurities), correct folding (FTIR, RF‐HPLC), monomer form (SEC‐HPLC), and active (bioactivity test). Also, the purification results revealed that expression at low temperature results in the efficient purification of the overproduced mecasermin with high quantity and quality.

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Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation

Cited by 9 publications

References 19 publications

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

Recombinant human G-CSF production as a protein based drug candidate for hematology and oncology

Efficient process development for high‐level production, purification, formulation, and characterization of recombinant mecasermin in Escherichia coli

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