Strategies for optimizing heterologous protein expression in Escherichia coli

Hannig, Gerhard; Makrides, Savvas C.

doi:10.1016/s0167-7799(97)01155-4

Cited by 409 publications

(194 citation statements)

References 30 publications

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“…Third, promoters should be capable of induction gene (Hannig and Makrides 1998). Besides, the strength of different promoters is determined by the relative frequency of transcription initiation.…”

Section: Discussionmentioning

confidence: 99%

“…3), were designed where the rare codons were replaced by frequently used ones in E. coli according to the codon usage table of E. coli (http://www.kazusa.or.jp/codon/). To increase the translation termination signal in E. coli, the original stop codon TGA was replaced by two stop codons in series, TAATGA, which is the most efficient translational termination sequence in E. coli (Hannig and Makrides 1998).…”

Section: Hd5 (Genbank Accession No M97925) Consists Of 94 Amino Acidsmentioning

confidence: 99%

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Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

Seo¹,

Weibel

Wehkamp

et al. 2012

International Journal of Medical Microbiology

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Section: Discussionmentioning

confidence: 99%

Section: Hd5 (Genbank Accession No M97925) Consists Of 94 Amino Acidsmentioning

confidence: 99%

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

Seo¹,

Weibel

Wehkamp

et al. 2012

International Journal of Medical Microbiology

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“…Therefore, 2 cDNAs of the mature IL-6 and IL-2 of giant panda were linked and cloned into E. coli expression plasmid pET-32a. Protein expression using E. coli is a widely used system to make proteins in large quantities (Hannig and Makrides, 1998;Sorensen and Mortensen, 2005). Like many other recombinant proteins, IL-6/IL-2 forms inclusion bodies when expressed in E. coli.…”

Section: Discussionmentioning

confidence: 99%

Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein

Nian²,

Ji³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. To construct a fusion cytokine protein with more and stronger bioactivities to enhance the immunity of the cytokine alone, we expressed interleukin (IL)-6/(IL)-2 from giant panda (Ailuropoda melanoleuca) in Escherichia coli as a 59.4-kDa fusion protein. Subsequently, the inclusion bodies were solubilized with 8 M urea and applied onto a Ni-nitrilotriacetic acid column. The final production of IL-6/IL-2 reached 6 mg/L in soluble form, and the purified final product was >96% pure. In Western blot assays, the recombinant IL-6/IL-2 was recognized by polyclonal antibodies against IL-6 and IL-2 of giant panda. The results demonstrated that the protein mixture contained correctly folded IL-2 and IL-6 proteins. A 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay demonstrated that IL-6/IL-2 can promote lymphocyte proliferation and differentiation. These data suggest that the fusion protein could be used to develop a novel immunoadjuvant to enhance the immunity of animals against infectious diseases.

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“…The pET22b vector carries an N-terminal pelB signal sequence for potential periplasmic localization and C-terminal His-Tag sequence, which is a pBR322-based plasmid and consists of the T7 promoter, the lac operator and lac repressor, which is encoded by the lacIq gene, and simultaneously, pET22b confers ampicillin resistance to the host (Studier and Moffatt, 1986;Studier et al, 1990). The most ordinary inducer of expression systems is IPTG (Hannig and Makrides, 1998). The lac repressor could bind to the lac operator to repress transcription, in the absence of the inducer IPTG.…”

Section: Hgp44 From P Gingivalis Inducing Huvecsmentioning

confidence: 99%

Study of Hgp44 from Porphyromonas gingivalis on inducing HUVECs to secrete IL-6 and IL-8

et al. 2014

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ABSTRACT. The aim of this study was to clone, express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis and purify the protein. Furthermore, the effect of Hgp44 from P. gingivalis on inducing HUVECs to secrete IL-6 and IL-8 was evaluated. The Hgp44 gene fragment was amplified by polymerase chain reaction, and then inserted into the cloning vector pMD18-T and linked with a prokaryotic expression vector pET22b to construct the recombinant expression plasmid pET22b-Hgp44. Fusion protein expression was induced by IPTG, and it was purified by immobilized metal-chelating affinity chromatography (IMAC) using an Ni 2+ matrix column. HUVECs were cultured in vitro and different concentrations of Hgp44 were added to confluent HUVEC monolayers and incubated for 2, 8 and 24 h. We extracted the supernatants and then used ELISA kits to test the changes in IL-6 and IL-8 levels. Finally, a 1100-bp fragment Hgp44 from P. gingivalis inducing HUVECs was successfully amplified, and the expression of the fusion protein was examined by SDS-PAGE and Western blot analysis, and the data showed that the protein was 44 kDa in size and expressed mostly in the form of inclusion bodies. The purification of the fusion protein was achieved using Ni 2+ affinity chromatography. About 3.5 mg/L fusion protein was obtained. Hgp44 could induce HUVECs to secrete IL-6 and IL-8 levels, which were remarkably increased. In a word, Hgp44 was successfully expressed in a prokaryotic expression system and purified by IMAC using the Ni 2+ matrix column. The effect of Hgp44 in inducing HUVECs to secrete IL-6 and IL-8 was demonstrated.

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Strategies for optimizing heterologous protein expression in Escherichia coli

Cited by 409 publications

References 30 publications

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein

Study of Hgp44 from Porphyromonas gingivalis on inducing HUVECs to secrete IL-6 and IL-8

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