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 incr...

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Autores principales:	Lei Zou, Sha Li, Nan Li, Shi-Long Ruan, Jing Chen, Jing Wu, Dazhong Yan, Hong-Jun Chao
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	fusion tag heterogenous protein solubility expression protocatechuate 3,4-dioxygenase solubility peptide Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/97885166f7654db8b1cbffc75608b29b
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spelling	oai:doaj.org-article:97885166f7654db8b1cbffc75608b29b2021-12-01T13:55:29ZThe Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli1664-302X10.3389/fmicb.2021.779541https://doaj.org/article/97885166f7654db8b1cbffc75608b29b2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.779541/fullhttps://doaj.org/toc/1664-302XEscherichia 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.Lei ZouSha LiNan LiShi-Long RuanJing ChenJing WuDazhong YanHong-Jun ChaoFrontiers Media S.A.articlefusion tagheterogenous proteinsolubility expressionprotocatechuate 3,4-dioxygenasesolubility peptideMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	fusion tag heterogenous protein solubility expression protocatechuate 3,4-dioxygenase solubility peptide Microbiology QR1-502
spellingShingle	fusion tag heterogenous protein solubility expression protocatechuate 3,4-dioxygenase solubility peptide Microbiology QR1-502 Lei Zou Sha Li Nan Li Shi-Long Ruan Jing Chen Jing Wu Dazhong Yan Hong-Jun Chao The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli
description	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.
format	article
author	Lei Zou Sha Li Nan Li Shi-Long Ruan Jing Chen Jing Wu Dazhong Yan Hong-Jun Chao
author_facet	Lei Zou Sha Li Nan Li Shi-Long Ruan Jing Chen Jing Wu Dazhong Yan Hong-Jun Chao
author_sort	Lei Zou
title	The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli
title_short	The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli
title_full	The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli
title_fullStr	The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli
title_full_unstemmed	The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli
title_sort	protocatechuate 3,4-dioxygenase solubility (pcds) tag enhances the expression and solubility of heterogenous proteins in escherichia coli
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/97885166f7654db8b1cbffc75608b29b
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The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

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