scholarly journals The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

2020 ◽  
Vol 21 (2) ◽  
pp. 576 ◽  
Author(s):  
Luciana Gomes ◽  
Gabriel Monteiro ◽  
Filipe Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
Chemical Induction ◽  
T7 Promoter ◽  
Iptg Induction ◽  
The Impact

This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms

2020 ◽  
Author(s):  
Luciana C. Gomes ◽  
Gabriel A. Monteiro ◽  
Filipe J. Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Heterologous Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
The Impact

<p><em>Escherichia coli</em> biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.</p> <p>In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from <em>E. coli</em> biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of <em>E. coli</em> JM109(DE3) transformed with a plasmid containing a T7 promoter.</p> <p>It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].</p> <p>It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.</p> <p> </p> <p> </p> <p>References:</p> <p>[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in <em>Escherichia coli</em> biofilms: A non-conventional form of high cell density cultivation. <em>Process Biochemistry, 57, 1-8</em>. https://doi.org/10.1016/j.procbio.2017.03.018</p> <p>[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by <em>Escherichia coli</em> Biofilms. <em>International Journal of Molecular Sciences, 21(2), 576</em>. https://doi.org/10.3390/ijms21020576</p>

scholarly journals The Influence of Nutrient Medium Composition on Escherichia coli Biofilm Development and Heterologous Protein Expression

2021 ◽  
Vol 11 (18) ◽  
pp. 8667
Author(s):  
Alexandra Soares ◽  
Luciana C. Gomes ◽  
Gabriel A. Monteiro ◽  
Filipe J. Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Culture Media ◽  
Plasmid Stability ◽  
Epifluorescence Microscopy ◽  
Egfp Expression ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
Biofilm Development

In the present study, the effects of different nutrient media on the development of Escherichia coli biofilms and the production of a heterologous protein were examined. E. coli JM109(DE3) cells transformed with pFM23 plasmid carrying the gene for enhanced green fluorescent protein (eGFP) expression were used. Cells were grown in two different culture media, Lysogenic Broth (LB) and M9ZB, in a flow cell system for 10 days. Epifluorescence microscopy, fluorimetry, and a high-performance liquid chromatography (HPLC) method based on hydrophobic interaction chromatography (HIC) were used to assess bacterial growth, plasmid copy number (PCN), and eGFP production in both planktonic and biofilm cells. The results showed that biofilm development was favored in M9ZB medium when compared with LB. However, the number of eGFP-expressing cells was higher in LB for both planktonic and sessile states (two-fold and seven-fold, respectively). In addition, the PCN in biofilm cells was slightly higher when using LB medium (on average, 29 plasmids per cell versus 20 plasmids per cell in M9ZB), and higher plasmid stability was observed in biofilms formed in LB compared to their planktonic counterparts. Hence, E. coli biofilms grown in LB enhanced both plasmid stability and capacity to produce the model heterologous protein when compared to M9ZB.

scholarly journals Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12497
Author(s):  
Fei Shang ◽  
Hui Wang ◽  
Dan Zhang ◽  
Wenhui Wang ◽  
Jiangliu Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Promoter Regions ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Pet Expression System

Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

scholarly journals The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Zou ◽  
Sha Li ◽  
Nan Li ◽  
Shi-Long Ruan ◽  
Jing Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Yellow Fluorescent Protein ◽  
Fluorescence Emission ◽  
Soluble Expression ◽  
Heterologous Protein Expression ◽  
E Coli

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression

2007 ◽  
Vol 1774 (12) ◽  
pp. 1536-1543 ◽  
Author(s):  
Kyung-Yeon Han ◽  
Hyuk-Seong Seo ◽  
Jong-Am Song ◽  
Keum-Young Ahn ◽  
Jin-Seung Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Transport Proteins ◽  
Heterologous Protein Expression

EVALUATION OF HETEROLOGOUS PROTEIN EXPRESSION AT DIFFERENT CONCENTRATIONS OF MGSO4 AND IPTG IN ESCHERICHIA COLI W110

2019 ◽  
pp. 111-118
Author(s):  
Yago Queiroz dos Santos ◽  
Gabriella Silva Campos Carelli ◽  
Bruno Oliveira de Veras ◽  
Joelton Igor Oliveira da Cruz ◽  
Geovanna Maria Medeiros Moura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression

FT-IR study of heterologous protein expression in recombinant Escherichia coli strains

2003 ◽  
Vol 1624 (1-3) ◽  
pp. 6-10 ◽  
Author(s):  
Diletta Ami ◽  
Loredana Bonecchi ◽  
Simona Calı̀ ◽  
Gaetano Orsini ◽  
Giancarlo Tonon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Recombinant Escherichia Coli ◽  
Heterologous Protein Expression ◽  
Ft Ir ◽  
Ir Study

Engineering glycolysis branch pathways of Escherichia coli to improve heterologous protein expression

2014 ◽  
Vol 49 (12) ◽  
pp. 2063-2070
Author(s):  
Xian-zhong Chen ◽  
Ying Xia ◽  
Wei Shen ◽  
You Fan ◽  
Algasan Govender ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression

scholarly journals A self-inducible heterologous protein expression system in Escherichia coli

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
L. Briand ◽  
G. Marcion ◽  
A. Kriznik ◽  
J. M. Heydel ◽  
Y. Artur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Protein Expression System
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