Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli

2017 ◽  
Vol 6 (4) ◽  
pp. 710-720 ◽  
Author(s):  
Jacob A. Englaender ◽  
J. Andrew Jones ◽  
Brady F. Cress ◽  
Thomas E. Kuhlman ◽  
Robert J. Linhardt ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
Genomic Integration

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

protocols.io ◽  
2019 ◽  
Author(s):  
Diep R ◽  
Timothy Rhodes ◽  
Nay Chi ◽  
Estee E ◽  
Kai Xun
Keyword(s):  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
E Coli

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

protocols.io ◽  
2020 ◽  
Author(s):  
Diep Ganguly ◽  
Timothy Rhodes ◽  
Nay Chi ◽  
Estee E ◽  
Kai Xun
Keyword(s):  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
E Coli

scholarly journals Heterologous protein expression in E. coli v2 (protocols.io.7x5hpq6)

protocols.io ◽  
2019 ◽  
Author(s):  
Diep R ◽  
Timothy Rhodes ◽  
Nay Chi ◽  
Estee E ◽  
Kai Xun
Keyword(s):  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
E Coli

scholarly journals Heterologous protein expression in E. coli v4 (protocols.io.95qh85w)

protocols.io ◽  
2019 ◽  
Author(s):  
Diep R ◽  
Timothy Rhodes ◽  
Nay Chi ◽  
Estee E ◽  
Kai Xun
Keyword(s):  
Protein Expression ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
E Coli

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.

Heterologous protein expression using a novel stress-responsive protein of E. coli RpoA as fusion expression partner

2007 ◽  
Vol 41 (6-7) ◽  
pp. 859-866 ◽  
Author(s):  
Keum-Young Ahn ◽  
Jong-Am Song ◽  
Kyung-Yeon Han ◽  
Jin-Seung Park ◽  
Hyuk-Seong Seo ◽  
...  
Keyword(s):  
Protein Expression ◽  
Heterologous Protein ◽  
Fusion Expression ◽  
Heterologous Protein Expression ◽  
E Coli

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

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.

Overexpression of the regulatory subunit of protein kinase A increases heterologous protein expression in Pichia pastoris

2020 ◽  
Vol 42 (12) ◽  
pp. 2685-2692 ◽  
Author(s):  
Xihao Liao ◽  
Wenyang Lin ◽  
Nanzhu Chen ◽  
Lu Li ◽  
Dafu Huang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Pichia Pastoris ◽  
Protein Expression ◽  
Protein Kinase A ◽  
Heterologous Protein ◽  
Regulatory Subunit ◽  
Heterologous Protein Expression ◽  
Kinase A
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