Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in <i>Escherichia coli</i>

Saito, Yoshimasa; Ishii, Yoshinori; Sasaki, Hirokazu; Hayashi, Masahito; Fujimura, Takao; Imai, Yuko; Nakamura, Sachiko; Suzuki, Shingo; Notani, Joji

doi:10.1021/bp00029a004

Cited by 8 publications

(1 citation statement)

References 37 publications

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“…The inclusion bodies have two main advantages of being mostly composed of recombinant proteins and easy isolation from the cell debris ( 12 , 26 ). However, inclusion bodies are insoluble and mainly inactive ( 12 , 27 , 28 ); thus the main problem in purification process of inclusion bodies is optimization of the refolding and renaturation states by prevention of the formation of inactive aggregates.…”

Section: Discussionmentioning

confidence: 99%

Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter

Shafiee

Moazen

Rabbani

et al. 2015

Jundishapur J Nat Pharm Prod

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Background:Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. Objectives: This study aimed at expression of reteplase in optimum condition. In this study, the reteplase gene was cloned and expressed in Escherichia coli top 10 as a suitable host cell and its expression was optimized. Materials and Methods: The recombinant plasmid, pET15b/reteplase was digested by NcoI and BamHI restriction enzymes; while pBAD/ gIIIA vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation of E. coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. Results: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of reteplase gene regarding the expression of reteplase. Maximum production of this enzyme was obtained under the following condition: 0.0002% L-arabinose at 37°C for 2 hours incubation. The purified protein was detected on SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis) as a 66 kDa band. The concentration of t-PA standard was 1 unit which is equal to 12 µg/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. Conclusions: Reteplase was expressed in E. coli Top 10 after activation of pBAD/gIIIA promoter region by arabinose and optimized.

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Section: Discussionmentioning

confidence: 99%

Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter

Shafiee

Moazen

Rabbani

et al. 2015

Jundishapur J Nat Pharm Prod

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Impact of oxygen supply on rtPA expression in Escherichia coli BL21 (DE3): ammonia effects

Wang

Wei

2009

Appl Microbiol Biotechnol

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In shake flasks, good oxygen supply tended to decrease rtPA expression in media containing only yeast extract and tryptone, while oxygen limitation would increase rtPA synthesis in the same medium. Our data showed that though the drop of rtPA expression in the 20-ml cultures of LBG or 2YTG was accompanied with a severe acetate accumulation, it was actually caused by low ammonia. The rtPA expression level could be significantly improved by increasing culture ammonium ion up to 500 mM. The effects of exogenous high ammonia on cell growth and rtPA expression were further examined in shake flasks and a 4-l fermentor. The high ammonia had no significant impact on cell growth and oxygen respiratory activity but significantly depressed the activities of glutamine synthetase/glutamate synthase and glutamate dehydrogenase, suggesting that ammonium ion as a nitrogen source improved the protein expression by mediating ammonia-assimilating enzymes. We thus propose in our work that E. coli cells, which were grown to a certain density to produce rtPA, would undergo nitrogen starvation under the low ammonia conditions even when the organic nitrogen sources remained abundant. The scale-up of rtPA production from shake flasks to fermentors could be readily achieved in the media containing rich ammonium ion.

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From shake flasks to bioreactors: survival of E. coli cells harboring pGST–hPTH through auto-induction by controlling initial content of yeast extract

Jia

Cheng

Wang

et al. 2011

Appl Microbiol Biotechnol

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A high content of yeast extract in complex media can cause auto-induction of phage T7 RNA polymerase and the consequent expression of recombinant protein in Escherichia coli BL21(DE3) during long-term cultivation. Our study demonstrated that the auto-induction of recombinant protein varied in different vectors harboring heterologous genes. Trx, GST, and their fusion proteins such as GST-human parathyroid hormone (hPTH), expressed by pET32a (+), were easily auto-induced by media containing a high content of yeast extract; however, rtPA was not easily auto-induced when using pET22b (+), although both pET systems were under the control of T7lac promoter. Furthermore, the auto-induction of GST-hPTH may start within 1-2 h after inoculation in bioreactors, which is a deficiency in the scale-up from shake flasks to bioreactors. Our results indicated that too much yeast extract in bioreactor cultivations may be responsible for the early auto-induction of target proteins and consequent loss of cell viability and plasmid instability. To achieve a satisfactory yield, host cells with both high cell viability and plasmid stability were necessary for the starter cultures in shake flasks and pre-induction cultures in bioreactors. This could be achieved simply by controlling the initial content of yeast extract and its subsequent supplementation.

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Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

Cited by 8 publications

References 37 publications

Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter

Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter

Impact of oxygen supply on rtPA expression in Escherichia coli BL21 (DE3): ammonia effects

From shake flasks to bioreactors: survival of E. coli cells harboring pGST–hPTH through auto-induction by controlling initial content of yeast extract

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