Effect of Preinduction Specific Growth Rate on Secretion of Granulocyte Macrophage Colony Stimulating Factor by <i>Escherichia coli</i>

Curless, Craig; Pope, J.; Loredo, Lawrence; Tsai, Larry B.

doi:10.1021/bp00029a003

Cited by 10 publications

(6 citation statements)

References 30 publications

(20 reference statements)

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“…A solution to prohibit the formation of inclusion bodies is to manipulate factors such as plasmid copy number, inducer concentration, and incubation time and temperature [34]. In this regard, bioprocess strategies for optimization of induction time [35], inducer availability [36], and pre-induction specific growth rate [37] have been recommended. It is worth mentioning that our further attempts towards the use of the modified BLA-derived signal peptides showed that an optimized concentration of lactose might improve the periplasmic expression level of rhGH (unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli

Jeiranikhameneh¹,

Moshiri²,

Falasafi³

et al. 2017

Journal of Microbiology and Biotechnology

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The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in , differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

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Section: Discussionmentioning

confidence: 99%

Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli

Jeiranikhameneh¹,

Moshiri²,

Falasafi³

et al. 2017

Journal of Microbiology and Biotechnology

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“…The specific growth rate is an important parameter to optimize the production of active protein with recombinant Escherichia coli . Fast growth during production (1−3) or even before induction (4−6) accelerates recombinant protein synthesis. With some systems, however, high growth rates deteriorate product accumulation (7−9 and additional references in ref 10).…”

Section: Introductionmentioning

confidence: 99%

Optimized Production of Active α-Glucosidase by Recombinant Escherichia coli. Evaluation of Processes Using in Vivo Reactivation from Inclusion Bodies

Ha¹,

Hoffmann²

2005

Biotechnol. Prog.

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During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.

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“…In both continuous and fed‐batch cultures the change of medium feed rate has been reported to be of great importance in controlling the specific growth rate [4], whereas in batch‐mode operation, its control was brought about by the change in medium composition [5]. Studies have been done to investigate the effect of both pre‐ [6–8] and post‐induction [5,9,10] specific growth rates of E. coli on recombinant protein synthesis. An increase in the specific growth rate of recombinant E. coli has been shown to result in a decrease in the expression level of the heterologous proteins in several recombinant systems [11,12], suggesting that maintenance of low specific growth rates in the production phase is required.…”

Section: Introductionmentioning

confidence: 99%

Effect of specific production rate of recombinant protein on multimerization of plasmid vector and gene expression level

Saraswat

Kim

Lee

et al. 1999

FEMS Microbiology Letters

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In fed-batch cultures of recombinant Escherichia coli BL21(DE3)[pT7-G3IL2] at high cell concentration, the post-induction specific growth rate was carefully regulated by controlled medium feed to maximize the synthesis level of recombinant fusion interleukin-2, G3.IL-2. A maximum concentration of G3.IL-2 (11.25 g l(-1)) was achieved in the induced recombinant culture growing at the rate of 0.056 h(-1). A steep decrease in the expression level of G3.IL-2 was observed at the post-induction specific growth rates higher than its optimal value (0.056 h(-1)). In the induced recombinant cultures, plasmid multimerization was observed and highly dependent on specific growth and production rate: a higher post-induction specific growth rate and an increased specific production rate tended to significantly promote it much further. Moreover, plasmid stability was found to decrease rapidly in a faster growing culture.

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Effect of Preinduction Specific Growth Rate on Secretion of Granulocyte Macrophage Colony Stimulating Factor by Escherichia coli

Cited by 10 publications

References 30 publications

Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli

Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli

Optimized Production of Active α-Glucosidase by Recombinant Escherichia coli. Evaluation of Processes Using in Vivo Reactivation from Inclusion Bodies

Effect of specific production rate of recombinant protein on multimerization of plasmid vector and gene expression level

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