Errors of heterologous protein expression

Kurland, C. G.; Gallant, Jonathan

doi:10.1016/s0958-1669(96)80050-4

Cited by 151 publications

(90 citation statements)

References 40 publications

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“…Over-expression of recombinant protein in E. coli may be severely diminished if the codes of the ORF for the protein use "rare" codons that are infrequently used by E. coli (8)(9)(10)(11)(12). According to the online (http://nihserver.mbi.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Dai

Zhang

et al. 2009

Cell Mol Immunol

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HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37 o C. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni 2+ -affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues. Cellular & Molecular Immunology. 2009; 6(4):261-268.

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Section: Discussionmentioning

confidence: 99%

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Dai

Zhang

et al. 2009

Cell Mol Immunol

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“…This could be attributed to misfolded proteins in E. coli compared to COS-7 cells, although the use of R. gami bacterial cells was intended to enhance properly folded and functional proteins, which is achieved by increasing the formation of disulfide bonds in the E. coli cytoplasm 65 and providing rare codon tRNAs compensating for insufficient levels in the E. coli system. 66 Further optimization of culture conditions and/or extraction techniques needs to be carried out to improve the efficiency of production.…”

Section: Discussionmentioning

confidence: 99%

Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose levels in db/db mice

et al. 2006

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Glucagon-like peptide (GLP-1), a major physiological incretin, plays numerous important roles in modulating blood glucose homeostasis and has been proposed for the treatment of type 2 diabetes. The major obstacles for using native GLP-1 as a therapeutic agent are that it must be delivered by a parenteral route and has a short half-life. In an attempt to develop a strategy to prolong the physiological t 1/2 and enhance the potency of GLP-1, a fusion protein consisting of active human GLP-1 and mouse IgG 1 heavy chain constant regions (GLP-1/Fc) was generated. A plasmid encoding an IgK leader peptide-driven secretable fusion protein of the active GLP-1 and IgG 1 -Fc was constructed for mammalian expression. This plasmid allows for expression of bivalent GLP-1 peptide ligands as a result of IgG-Fc homodimerization. In vitro studies employing purified GLP-1/ Fc indicate that the fusion protein is functional and elevates cAMP levels in insulin-secreting INS-1 cells. In addition, it stimulates insulin secretion in a glucose concentrationdependent manner. Intramuscular gene transfer of the plasmid in db/db mice demonstrated that expression of the GLP-1/Fc peptide normalizes glucose tolerance by enhancing insulin secretion and suppressing glucagon release. This strategy of using a bivalent GLP-1/Fc fusion protein as a therapeutic agent is a novel approach for the treatment of diabetes.

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“…Escherichia coli is commonly used for expression of high levels of heterologous proteins, especially in the commercial setting. However this organism has a marked bias in codon usage, 18 and thus the FAVAC-1A gene sequence was adjusted to optimize to E. coli's preferred usage.…”

Section: Introductionmentioning

confidence: 99%

Development, Characterization and Immunogenicity of a Multi-Stage, MultivalentPlasmodium falciparumVaccine Antigen (FALVAC-1A) Expressed inEscherichia coli

Zhou¹,

Todd²,

Wohlhueter³

et al. 2006

Human Vaccines

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A synthetic multistage, multi-epitope Plasmodium falciparum malaria antigen (FALVAC-1A) was designed and evaluated in silico, and then the gene was constructed and expressed in Escherichia coli. The FALVAC-1A protein was purified by inclusion body isolation, followed by affinity and ion exchange chromatography. Although FALVAC-1A was a synthetic antigen, it folded to a specific, but as yet incompletely defined, molecular conformation that was stable and comparable from lot to lot. When formulated with four different adjuvants, FALVAC-1A was highly immunogenic in rabbits, inducing not only ELISA reactivity to the cognate antigen and most of its component epitopes, but also in vitro activity against P. falciparum parasites as demonstrated by inhibition of sporozoite invasion, antibody dependent cellular inhibition and the immunofluorescence assay.

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Errors of heterologous protein expression

Cited by 151 publications

References 40 publications

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose levels in db/db mice

Development, Characterization and Immunogenicity of a Multi-Stage, MultivalentPlasmodium falciparumVaccine Antigen (FALVAC-1A) Expressed inEscherichia coli

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