N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae

Ojima-Kato, Teruyo; Nagai, Sonoko; Nakano, Hideo

doi:10.1016/j.jbiosc.2016.12.004

Cited by 49 publications

(49 citation statements)

References 33 publications

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“…In addition, it was found that the effect was more prominent in the presence of DsbC. Consistent with our previous study 24 , it was also found that addition of the SKIK tag increased protein synthesis of both the Hc and Lc in CFPS (Fig. 2b ).…”

Section: Resultssupporting

confidence: 91%

“…Most of the target proteins were produced in insoluble form, consistent with our previous study of the SKIK tag (Supplementary Fig. S2a ) 24 . Therefore, we tried refolding the insoluble Zipbody by the method used for scFv 28 , and successfully recovered > 80% of the insoluble proteins in a soluble form without optimization of the refolding process (Supplementary Fig.…”

Section: Discussionsupporting

confidence: 83%

“…Unless otherwise stated, mAbs produced in CFPS or E . coli were analysed by SDS-PAGE in reducing conditions and ELISA using our standard protocol described in reference 24 . In brief, proteins separated on SDS-PAGE were visualised by CBB staining, western blotting detecting tags using horseradish peroxidase (HRP)-conjugated antibodies (anti-FLAG tag-HRP [GeneTex, Irvine, CA], anti-His tag-HRP [Medical and Biological Laboratories, Nagoya, Japan], or anti-HA tag-HRP [Wako, Osaka, Japan]) and 1-Step™ TMB-Blotting Substrate Solution (Thermo, Waltham, MA), or fluorescent imaging.…”

Section: Methodsmentioning

confidence: 99%

“…The expression of Zipbody with an SKIK tag was carried out using E . coli SHuffle T7 Express as the host, grown in 50 mL LB medium containing 100 μg/mL ampicillin with 1 mM IPTG induction at 16 °C for 24 h, as described in our previous study 24 .…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, we recently found that the addition of a short peptide sequence tag Ser-Lys-Ile-Lys (SKIK) to the N-terminus of so-called ‘difficult-to-express proteins’ can dramatically improve their expression level, both in E . coli in vivo and in in vitro expression systems 24 .…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

Ojima-Kato

Nagai

Nakano

2017

Sci Rep

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We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named “Ecobody technology”. The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the VH and VL genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) ‘Zipbody’ as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (K D = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

show abstract

Section: Resultssupporting

confidence: 91%

Section: Discussionsupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

Ojima-Kato

Nagai

Nakano

2017

Sci Rep

Self Cite

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New tools for recombinant protein production in Escherichia coli: A 5‐year update

2019

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The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.

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Factors involved in heterologous expression of proteins in E. coli host

Pouresmaeil

Azizi-Dargahlou

2023

Arch Microbiol

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The production of recombinant proteins is one of the most significant achievements of biotechnology in the last century. These proteins are produced in the eukaryotic or prokaryotic heterologous hosts. By increasing the omics data especially related to different heterologous hosts as well as the presence of new amenable genetic engineering tools, we can artificially engineer heterologous hosts to produce recombinant proteins in sufficient quantities. Numerous recombinant proteins have been produced and applied in various industries, and the global recombinant proteins market size is expected to be cast to reach USD 2.4 billion by 2027. Therefore, identifying the weakness and strengths of heterologous hosts is critical to optimize the large-scale biosynthesis of recombinant proteins. E. coli is one of the popular hosts to produce recombinant proteins. Scientists reported some bottlenecks in this host, and due to the increasing demand for the production of recombinant proteins, there is an urgent need to improve this host. In this review, we first provide general information about the E. coli host and compare it with other hosts. In the next step, we describe the factors involved in the expression of the recombinant proteins in E. coli . Successful expression of recombinant proteins in E. coli requires a complete elucidation of these factors. Here, the characteristics of each factor will be fully described, and this information can help to improve the heterologous expression of recombinant proteins in E. coli .

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N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae

Cited by 49 publications

References 33 publications

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

New tools for recombinant protein production in Escherichia coli: A 5‐year update

Factors involved in heterologous expression of proteins in E. coli host

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