Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli

Nik-Pa, Nik Ida Mardiana; Sobri, Mohamad Farhan Mohamad; Abd‐Aziz, Suraini; Ibrahim, Mohamad Faizal; Bahrin, Ezyana Kamal; Alitheen, Noorjahan Banu; Ramli, Norhayati

doi:10.3390/ijms21113919

Cited by 15 publications

(5 citation statements)

References 42 publications

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“…Codon usage is critical to achieving adequate protein expression levels in E. coli [32], [33]. This technique can improve the production of various recombinant proteins expressed by E. coli [34], [35], [36]. Blastx analysis from the SEBsyn consensus sequence was showed that this sequence has specific hits with SEB protein sequences in the database with identities ranging about 97-100 %.…”

Section: Resultsmentioning

confidence: 99%

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Rodiansyah¹,

Tan²,

Nugrahapraja³

2023

Int. J. Adv. Sci. Eng. Inf. Technol.

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Staphylococcal enterotoxin B (SEB) from Staphylococcus aureus is a potential therapeutic agent for cancer. SEB can activate the immune response, which could induce apoptosis of various cancer cells. This study aimed to design and produce a recombinant SEB protein using Escherichia coli BL21(DE3) expression system. We designed and optimized the codon of the inserted gene for E. coli and then transformed it into E. coli BL21(DE3). The transformants were verified by selective media containing kanamycin followed by colony PCR with T7 promoter and T7 terminator primers and DNA sequencing. The results showed that the SEBsyn encoding gene could be synthesized and cloned into pET-28a(+) expression plasmid. This recombinant plasmid that carried the SEB encoding gene (pET-28a_SEBsyn) was successfully transformed into E. coli BL21(DE3). The amplicons of colony PCR visualized in 2% agarose gel showed that transformants carrying the recombinant plasmid had an inserted gene length of about 1 kb. Gene inserts sequence verification by DNA sequencing resulted in 1,148 bp sequence consensus. The blastx analysis showed that it had the best hit with enterotoxin B in the NCBI database (accession id CAC6284025.1). The recombinant SEB protein was also successfully overexpressed in E. coli BL21(DE3) with 0.1 mM IPTG. Our recombinant SEB protein was dominantly insoluble in the inclusion body; however, some SEB protein was expressed as soluble. The molecular weight of the target protein is about 29 kDa.

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Section: Resultsmentioning

confidence: 99%

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Rodiansyah¹,

Tan²,

Nugrahapraja³

2023

Int. J. Adv. Sci. Eng. Inf. Technol.

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“…We first evaded expression problems due to codon bias (incompatibility of codon usage between human sTNF-α gene and E. coli CFPS system) via computational optimization of the codon profile of the human sTNF-α gene as previously described for human sTNF-α and other recombinant proteins [ 7 , 22 , 36 , 47 , 48 ].…”

Section: Discussionmentioning

confidence: 99%

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha

El-Baky

El‐Fakharany

Sabry

et al. 2022

Biology

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Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

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“…The C-terminally attached His6-tag is the common design for a nity puri cation of the extracellularly produced proteins [6][7][8][9][10][11]. In a few cases, the His6-tag is incorporated between the target protein and Cterminal signal peptide [12], or fused to the N-terminus of the target protein [13,14], for purifying the protein secreted into the culture. The a nity tags including glutathione S-transferase (GST) as the Cterminal fusion partner, and maltose binding protein as the N-terminal carrier are occasionally used for rapid puri cation of the produced proteins of interest via extracellular production [15,16].…”

Section: Introductionmentioning

confidence: 99%

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Chen,

Li,

et al. 2024

Preprint

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Background Rapid and cost-effective purification of the target protein for extracellular production in Escherichia coli is still challenge. Previously, we identified that human annexin A1 as a N-terminal fusion tag for Ca2+-dependent phase transition to simply, rapidly and cost-effectively purify three fluorescent proteins including emerald green fluorescent protein (EmGFP), red fluorescent protein mCherry, and flavin-binding cyan-green fluorescent protein.Results When the phage lytic protein was induced later, the annexin A1 tagged EmGFP was leaked into the culture, but purification efficiency was relatively low. Pre-overexpression of Bacillus cereus phospholipase C facilitated intracellular production of the fusion protein, and purified fusion protein showed the purity higher than other two fluorescent protein fusions. Using the co-expression system, the elastin-like polypeptide (ELP) tagged three fluorescent proteins via extracellular production were also purified in revisable protein precipitation. The yield of the purified annexin A1 tagged protein was comparable to that of the purified ELP tagged one. The silica-binding peptide tagged annexin A1-EmGFP bound to silica particles, and the ELP tagged mCherry strongly bound to phenyl sepharose was efficient for column-dependent purification. The extracellular nine tobacco etch virus protease variants with the annexin A1 tag were purified and the cleavage activity was assayed. Using the purified protease variant with the highest activity, the purification tag was removed in solution, or by on-resin cleavage of the immobilized annexin A1 or ELP tagged EmGFP. The soluble annexin A1-EmGFP with the Bacillus amyloliquefaciens alpha-amylase signal peptide was poorly produced in Bacillus subtilis, and the fusion protein with the α-factor signal peptide was located in intracellular Pichia pastoris.Conclusions The annexin A1 or ELP fusions in the culture were purified by revise transition cycles. On-resin cleavage facilitated removal of the reagents for protein purification, and fusion tag. However, the annexin A1-EmGFP fused the correspondent signal peptides displayed poor secretion efficiency in B. subtilis and P. pastoris. The platform will be used for simply and cost-effectively purifying the target proteins with industrial and clinical values without cell disruption process, and rapidly testifying the activity of the engineered enzyme variants.

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Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli

Cited by 15 publications

References 42 publications

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

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