Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis

Le, Ngan Thi Phuong; Phan, Trang Thi Phuong; Phan, Hanh Thi Thu; Truong, Tuom Thi Tinh; Schumann, Wolfgang; Nguyen, Hoang Duc

doi:10.1016/j.btre.2022.e00754

Cited by 6 publications

(11 citation statements)

References 35 publications

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“…The number of cells collected was equivalent to the quantity found in 1 mL of culture with an OD 600 value of 2.4. The target proteins were analyzed by fluorescence and SDS‐PAGE, as previously described 7 27 …”

Section: Methodsmentioning

confidence: 99%

“…The effectiveness of recombinant protein expression in B. subtilis depends on many factors, such as optimization of B. subtilis strains (e.g., secretion pathway optimization, enhanced RNA and protein stability, improved cell growth, genome streamlining, engineering of transcriptional regulators, and the implementation of genome‐editing methodologies), 3,4 as well as the wide availability and efficiency of promoters 2 . Moreover, the use of fusion tags can affect the production levels of recombinant proteins 5–7 …”

Section: Introductionmentioning

confidence: 99%

“…Among three different His‐tag sequences, MGRS‐8xHis gave the highest expression level of GFP+ protein, which was 10 times lower compared to the control without His‐tag protein (pHT1066). For the low‐expression protein EGFP, the expression level increased up to 15 times 7 …”

Section: Introductionmentioning

confidence: 99%

“…2 Moreover, the use of fusion tags can affect the production levels of recombinant proteins. [5][6][7] Fusion tag, such as protein domains, entire proteins, or small peptides fused at the N-or C-terminus of the target proteins to enhance their solubility, expression level, and purification. 8 The fusion tag's position, sequence, and length can impact the fusion protein, including production levels, solubility, binding to ligandimmobilized metal affinity chromatography (IMAC), tendency to crystal formation, tertiary structure, and activity.…”

Section: Introductionmentioning

confidence: 99%

“…8 The fusion tag's position, sequence, and length can impact the fusion protein, including production levels, solubility, binding to ligandimmobilized metal affinity chromatography (IMAC), tendency to crystal formation, tertiary structure, and activity. [5][6][7] The utilization of fusion tags can increase the protein expression level and/or the solubility, helping to achieve natural folding or enhance the expression level for a low-expression protein. 9 Cell Biochem Funct.…”

Section: Introductionmentioning

confidence: 99%

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N‐terminal LysSN‐His‐tag improves the production of intracellular recombinant protein in Bacillus subtilis

Phan

Truong

et al. 2023

Cell Biochemistry & Function

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Choosing fusion tags to enhance the recombinant protein levels in the cytoplasm of Bacillus subtilis has been limited. Our previous study demonstrated that His‐tag at the N‐terminus could increase the expression levels of the low‐expression gene egfp, while significantly reducing the high‐expression genes gfp+ and bgaB in the cytoplasm of B. subtilis. In this study, we aimed to prove the potential of a fusion tag, the combination of the N‐terminal domain of B. subtilis lysyl tRNA synthetase (LysSN) and His‐tag with varying numbers of histidine (6xHis, 8xHis, 10xHis) by investigating their effects on the expression levels of egfp, gfp+ and bgaB in B. subtilis. For the low‐expression gene, LysSN‐xHis‐tag could enhance the fluorescent intensity of EGFP 23.5 times higher than EGFP without a fusion tag, and 1.5 times higher than that fused with only His‐tag. For high‐expression genes, the expression level of BgaB and GFP+ was 2.9 and 12.5 times higher than that of His‐tag, respectively. The number of histidines in LysSN‐xHis‐tag did not influence the expression levels of the high‐expression genes but affected the expression levels of the low‐expression gene.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

N‐terminal LysSN‐His‐tag improves the production of intracellular recombinant protein in Bacillus subtilis

Phan

Truong

et al. 2023

Cell Biochemistry & Function

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A novel epitope tag from rabies virus has versatile in vitro applications

Zhang

Wang

et al. 2023

Appl Microbiol Biotechnol

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Cloning and expression of recombinant human superoxide dismutase 1 (hSOD1) in Bacillus subtilis 1012

Yin

Wang

et al. 2022

Biotechnol Lett

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As an important member of the antioxidant defense system in vivo, human Cu, Zn superoxide dismutase (hSOD1) has become a potential therapeutic agent against host diseases, and new strategies were developed to achieve high-yield SOD1 by using e cient production systems, like E. coli, yeast, and other eukaryotic expression systems. However, the above systems have their own limitations, such as the formation of inclusion bodies, endotoxins production, and low secretion e ciency. In this study, the hSOD1 coding sequence gene was ampli ed from human HepG2 cells, cloned into a pHT43-His expression vector, and transformed into Bacillus subtilis system. After the optimization with different media, temperatures, and inducer (IPTG) concentrations, the recombinant hSOD1 was successfully expressed in Bacillus subtilis 1012. The results show that hSOD1 was produced as a soluble form in Super rich medium with 0.2 mM of IPTG at 37°C after the induction for 24 h. Besides, 20 g/L of lactose also displayed the same inductive effect on hSOD1 expression as that by 0.2 mM of IPTG. Finally, hSOD1 was e ciently puri ed by nickel a nity chromatography, and the speci c activity of hSOD1 was determined to be 1625 U/mg in the presence of 800 µM of Cu 2+ and 20 µM of Zn 2+ . Collectively, Bacillus subtilis 1012 can not only overcome the shortcomings of E. coli and yeast expression systems, but also be directly used for disease treatment as a healthy probiotic. These advantages provided a possibility for the application of oral engineering bacteria 1012-hSOD1 to the clinical treatment in the future.

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Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis

Cited by 6 publications

References 35 publications

N‐terminal LysSN‐His‐tag improves the production of intracellular recombinant protein in Bacillus subtilis

N‐terminal LysSN‐His‐tag improves the production of intracellular recombinant protein in Bacillus subtilis

A novel epitope tag from rabies virus has versatile in vitro applications

Cloning and expression of recombinant human superoxide dismutase 1 (hSOD1) in Bacillus subtilis 1012

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