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

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A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-021-01725-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Hyunjun Ko ◽

Minsik Kang ◽

Mi-Jin Kim ◽

Jiyeon Yi ◽

Jin Kang ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Inclusion Bodies ◽

Heterologous Protein ◽

Soluble Expression ◽

Carbohydrate Binding ◽

Heterologous Protein Expression ◽

Carbohydrate Binding Module ◽

Fusion Tag ◽

E Coli

Abstract Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

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

Applied Sciences ◽

10.3390/app11188667 ◽

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.

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Strategies for optimizing heterologous protein expression in Escherichia coli

Trends in Biotechnology ◽

10.1016/s0167-7799(97)01155-4 ◽

1998 ◽

Vol 16 (2) ◽

pp. 54-60 ◽

Cited By ~ 295

Author(s):

Gerhard Hannig ◽

Savvas C Makrides

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Heterologous Protein ◽

Heterologous Protein Expression ◽

Expression In Escherichia Coli

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

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2007.09.012 ◽

2007 ◽

Vol 1774 (12) ◽

pp. 1536-1543 ◽

Cited By ~ 15

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

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Heterologous protein expression in E. coli v3 (protocols.io.9vgh63w)

protocols.io ◽

10.17504/protocols.io.9vgh63w ◽

2019 ◽

Author(s):

Diep R ◽

Timothy Rhodes ◽

Nay Chi ◽

Estee E ◽

Kai Xun

Keyword(s):

Protein Expression ◽

Heterologous Protein ◽

Heterologous Protein Expression ◽

E Coli

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Heterologous protein expression in E. coli v5 (protocols.io.bdjti4nn)

protocols.io ◽

10.17504/protocols.io.bdjti4nn ◽

2020 ◽

Author(s):

Diep Ganguly ◽

Timothy Rhodes ◽

Nay Chi ◽

Estee E ◽

Kai Xun

Keyword(s):

Protein Expression ◽

Heterologous Protein ◽

Heterologous Protein Expression ◽

E Coli

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

Conceitos Básicos da Genética ◽

10.22533/at.ed.21419210610 ◽

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

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

International Journal of Molecular Sciences ◽

10.3390/ijms21020576 ◽

2020 ◽

Vol 21 (2) ◽

pp. 576 ◽

Cited By ~ 2

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.

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